Methods and devices for surgical pre-treatment

ABSTRACT

An apparatus includes multiple first reservoirs and multiple second reservoirs joined with a substrate. Selected ones of the multiple first reservoirs include a reducing agent, and first reservoir surfaces of selected ones of the multiple first reservoirs are proximate to a first substrate surface. Selected ones of the multiple second reservoirs include an oxidizing agent, and second reservoir surfaces of selected ones of the multiple second reservoirs are proximate to the first substrate surface.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. Provisional patentapplication Ser. No. 15/315,972, filed Dec. 2, 2016, which is a 371 ofPCT US/2015/34033, filed Jun. 3, 2015, which claims benefit under 35U.S.C. § 119(e) from United States Provisional Patent Application Ser.No.'s 62/007,295, filed Jun. 3, 2014; 62/012,006, filed Jun. 13, 2014;62/090,011, filed Dec. 10, 2014; 62/137,987, filed Mar. 25, 2015;62/138,041, filed Mar. 25, 2015; and 62/153,163, filed Apr. 27, 2015;the content of each of which is incorporated herein by reference in itsentirety.

FIELD

Biologic tissues and cells are affected by a unique electrical stimulus.Accordingly, apparatus and techniques for applying electric stimulus totissue have been developed to address a number of medical issues. Thepresent specification relates to methods and devices useful fortreatment of tissue prior to surgery or other disruption.

BACKGROUND

The skin is a natural barrier against infection; however any surgerythat causes a break in the skin can lead to a postoperative infection,often described as surgical site infections (SSTs). Following surgery,the chances of developing an SSI are about 1 to 3 percent. SS's areoften one or more of three types. A “superficial incisional” SSI occursin the area of the skin where the surgical incision was made. A “deepincisional” SSI occurs beneath the incision area in muscle tissue and infascia, as well as the tissue surrounding the muscles. An “organ” or“space” SSI can occur in any area of the body other than the skin,muscle, or fascia that was involved in the surgery, such as an organ orbetween organs.

Infections after surgery are often caused by microorganisms. The mostcommon of these include the bacteria Staphylococcus, Streptococcus, andPseudomonas. Microorganisms can infect a surgical wound through variousforms of contact, such as from the touch of a contaminated caregiver orsurgical instrument, through microorganisms in the air, or throughmicroorganisms that are already on or in the body. The degree of riskfor an SSI is linked to the type of surgery performed—risk factors forSS's include surgery that lasts more than two hours; advanced age,obesity, smoking, diabetes, and cancer.

SUMMARY

Embodiments disclosed herein include systems, devices, and methods forpre-treating surgical sites, for example using bioelectric devices thatcomprise a multi-array matrix of biocompatible microcells and a fluidsuch as a conductive fluid or cream, for example an antibacterial. Bypre-treating surgical sites, the presence of microorganisms surroundingthe surgery site can be limited or reduced. In addition, pre-treatmentof the surgery site can stimulate the healing process by increasing cellmigration, ATP production, and angiogenesis.

Embodiments disclosed herein comprise bioelectric devices that comprisea multi-array matrix of biocompatible microcells. Such matrices caninclude a first array comprising a pattern of microcells, for exampleformed from a first conductive solution, the solution including a metalspecies; and a second array comprising a pattern of microcells, forexample, formed from a second conductive solution, the solutionincluding a metal species capable of defining at least one voltaic cellfor spontaneously generating at least one electrical current with themetal species of the first array when said first and second arrays areintroduced to an electrolytic solution and said first and second arraysare not in physical contact with each other. Certain aspects utilize anexternal power source such as AC or DC power, or pulsed RF, or pulsedcurrent, such as high voltage pulsed current. In one embodiment, theelectrical energy is derived from the dissimilar metals creating abattery at each cell/cell interface, whereas those embodiments with anexternal power source can employ conductive electrodes in a spacedconfiguration to predetermine the electric field shape and strength.

Aspects disclosed herein include systems, devices, and methods forpreventing damage to, repairing, and rejuvenating teeth and gums, forexample using bioelectric devices that comprise a multi-array matrix ofbiocompatible microcells and a fluid such as a conductive fluid, orcream, or saliva.

Also disclosed herein are electroceutical fabrics.

Also disclosed herein are dressings for use with medical devices thatcan be inserted into the body, such as catheters.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a detailed plan view of an embodiment disclosed herein.

FIG. 2 is a detailed plan view of a pattern of applied electricalconductors in accordance with an embodiment disclosed herein.

FIG. 3 is an embodiment using the applied pattern of FIG. 2 .

FIG. 4 is a cross-section of FIG. 3 through line 3-3.

FIG. 5 is a detailed plan view of an alternate embodiment disclosedherein which includes fine lines of conductive metal solution connectingelectrodes.

FIG. 6 is a detailed plan view of another alternate embodiment having aline pattern and dot pattern.

FIG. 7 is a detailed plan view of yet another alternate embodimenthaving two line patterns.

FIG. 8 depicts alternate embodiments showing the location ofdiscontinuous regions as well as anchor regions of the system.

FIG. 9 depicts an embodiment showing a mask comprising a multi-arraymatrix of biocompatible microcells and means for securing the mask.

FIG. 10 depicts an embodiment for pre-treating prior to surgery on theback.

FIG. 11 depicts an embodiment disclosed herein for pre-treatment of asmall area. The embodiment includes a hydrated silica pack to maintainhydration of the device.

FIG. 12 depicts PROCELLERA® (an embodiment disclosed herein) output overtime. The graph shows volts plotted against time in hours.

FIG. 13 depicts an electroceutical fabric as described herein.

FIG. 14 depicts an electroceutical fabric as described herein.

FIG. 15 depicts an embodiment as disclosed for oral use.

FIG. 16 depicts a catheter dressing as disclosed herein.

FIG. 17 depicts a catheter dressing as disclosed herein.

FIG. 18(A) is an Energy Dispersive X-ray Spectroscopy (EDS) analysis ofAg/Zn BED (“bioelectric device”; refers to an embodiment as disclosedherein).

-   -   a. Scanning Electron Microscope (SEM) image;    -   b. Light Microscope Image;    -   c. Closer view of a golden dot and a grey dot in B respectively.    -   d. Closer view of a golden dot and a grey dot in B respectively.    -   e. EDS element map of zinc;    -   f. EDS element map of silver;    -   g. EDS element map of oxygen;    -   h. EDS element map of carbon. Scale bar A-B, E-H: 1 mm; C-D: 250        μm

18(B,C) Absorbance measurement on treating planktonic PAO1 culture withplacebo, Ag/Zn BED and placebo +Ag dressing; and CFU measurement.

18(D) Zone of inhibition with placebo, Ag/Zn BED and placebo +Agdressing.

FIG. 19 depicts Scanning Electron Microscope (SEM) images of in-vitroPseudomonas aeruginosa PAO1 biofilm treated with placebo, an embodimentdisclosed herein (“BED”), and placebo +Ag dressing. The BED treatedbiofilm shows a dramatic decrease in bacteria number.

FIG. 20 shows extracellular polysaccharide staining (EPS).

FIG. 21 shows live/dead staining. The green fluorescence indicates livePAO1 bacteria while the red fluorescence indicates dead bacteria.

FIG. 22 shows PAO1 staining.

FIG. 23 depicts real-time PCR to assess quorum sensing gene expression.

FIG. 24 shows electron paramagnetic (EPR) spectra using DEPMPO (aphosphorylated derivative of the widely used DMPO spin trap). Spinadduct generation upon exposure to disclosed embodiments for 40 minutesin PBS.

FIG. 25 depicts real-time PCR performed to assess mex gene expression up on treatment with Ag/Zn BED and 10 mM DTT.

FIG. 26 shows Glycerol-3-Phosphate Dehydrogenase (GPDH) enzyme activity.

-   -   a. OD was measured in the kinetic mode.    -   b. GPDH activity was calculated using the formula,        Glycerol-3-Phosphate dehydrogenase activity=B/(ΔT ×V) ×Dilution        Factor=nmol/min/ml, where:        -   B=NADH amount from Standard Curve (nmol). ΔT=reaction time            (min).        -   V=sample volume added into the reaction well (ml).

DETAILED DESCRIPTION

Embodiments disclosed herein include systems and devices that canprovide a low level electric field (LLEF) to a tissue or organism (thusa “LLEF system”) or, when brought into contact with an electricallyconducting material, can provide a low level electric micro-current(LLEC) to a tissue or organism (thus a “LLEC system”). Thus, inembodiments a LLEC system is a LLEF system that is in contact with anelectrically conducting material, for example a liquid material. Incertain embodiments, the micro-current or electric field can bemodulated, for example, to alter the duration, size, shape, field depth,duration, current, polarity, or voltage of the system. For example, itcan be desirable to employ an electric field of greater strength ordepth in an area where skin is thicker to achieve optimal treatment. Inembodiments the watt-density of the system can be modulated.

Definitions

“Activation gel” as used herein means a composition useful formaintaining a moist environment within and about the skin. Activationgels can be conductive. Activation gels can also be antibacterial.

“Affixing” as used herein can mean contacting a patient or tissue with adevice or system disclosed herein. In embodiments “affixing” can includethe use of straps, elastic, etc.

“Antibacterial agent” or “antibacterial” as used herein refers to anagent that interferes with the growth and reproduction of bacteria.Antibacterial agents are used to disinfect surfaces and eliminatepotentially harmful bacteria. Unlike antibiotics, they are not used asmedicines for humans or animals, but are found in products such assoaps, detergents, health and skincare products and household cleaners.Antibacterial agents may be divided into two groups according to theirspeed of action and residue production: The first group contains thosethat act rapidly to destroy bacteria, but quickly disappear (byevaporation or breakdown) and leave no active residue behind (referredto as non-residue-producing). Examples of this type are the alcohols,chlorine, peroxides, and aldehydes. The second group consists mostly ofnewer compounds that leave long-acting residues on the surface to bedisinfected and thus have a prolonged action (referred to asresidue-producing). Common examples of this group are triclosan,triclocarban, and benzalkonium chloride. As used herein, “antibacterialagent” includes sanitizers, disinfectants, and sterilizers.Antibacterials can be used to reduce the population of proliferation ofbacteria, for example Acinetobacter baumannii, Corynebacteriumamycolatum, Enterobacter aerogenes, Enterococcus faecalis, Escherichiacoli, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratia marcescens,Staphylococcus aureus, and Staphylococcus epidermidis, or the like.

“Applied” or “apply” as used herein refers to contacting a surface witha conductive material, for example printing, painting, or spraying aconductive ink on a surface. Alternatively, “applying” can meancontacting a patient or tissue or organism with a device or systemdisclosed herein.

“Catheter” as used herein refers to a thin tube made from medical gradematerials serving a broad range of functions. Catheters are medicaldevices that can be inserted in the body to treat diseases or perform asurgical procedure. Catheters can be inserted into a body cavity, duct,or vessel. Functionally, they allow drainage, administration of fluidsor gases, access by surgical instruments, and also perform a widevariety of other tasks depending on the type of catheter. The process ofinserting a catheter is catheterization. In most uses, catheter is athin, flexible tube (“soft” catheter) though catheters are available invarying levels of stiffness depending on the application.

“Conductive material” as used herein refers to an object or type ofmaterial which permits the flow of electric charges in one or moredirections. Conductive materials can include solids such as metals orcarbon, or liquids such as conductive metal solutions and conductivegels. Conductive materials can be applied to form at least one matrix.Conductive liquids can dry, cure, or harden after application to form asolid material.

“Discontinuous region” as used herein refers to a “void” in a materialsuch as a hole, slot, or the like. The term can mean any void in thematerial though typically the void is of a regular shape. A void in thematerial can be entirely within the perimeter of a material or it canextend to the perimeter of a material.

“Disruption” or “disrupted space” as used herein refers to any break,tear, void, cut, scratch, bit, sore, wound, gum recession, open cavity,gum flap, void in gums between tooth and gum, dental extraction, cleftpalate, opening caused by surgery, or the like.

“Dots” as used herein refers to discrete deposits of dissimilarreservoirs that can function as at least one battery cell. The term canrefer to a deposit of any suitable size or shape, such as squares,circles, triangles, lines, etc. The term can be used synonymously with,microcells, etc.

“Electrode” refers to similar or dissimilar conductive materials. Inembodiments utilizing an external power source the electrodes cancomprise similar conductive materials. In embodiments that do not use anexternal power source, the electrodes can comprise dissimilar conductivematerials that can define an anode and a cathode.

“Expandable” as used herein refers to the ability to stretch whileretaining structural integrity and not tearing. The term can refer tosolid regions as well as discontinuous or void regions; solid regions aswell as void regions can stretch or expand.

“Galvanic cell” as used herein refers to an electrochemical cell with apositive cell potential, which can allow chemical energy to be convertedinto electrical energy. More particularly, a galvanic cell can include afirst reservoir serving as an anode and a second, dissimilar reservoirserving as a cathode. Each galvanic cell can store chemical potentialenergy. When a conductive material is located proximate to a cell suchthat the material can provide electrical and/or ionic communicationbetween the cell elements the chemical potential energy can be releasedas electrical energy. Accordingly, each set of adjacent, dissimilarreservoirs can function as a single-cell battery, and the distributionof multiple sets of adjacent, dissimilar reservoirs within the apparatuscan function as a field of single-cell batteries, which in the aggregateforms a multiple-cell battery distributed across a surface. Inembodiments utilizing an external power source the galvanic cell cancomprise electrodes connected to an external power source, for example abattery or other power source. In embodiments that areexternally-powered, the electrodes need not comprise dissimilarmaterials, as the external power source can define the anode andcathode. In certain externally powered embodiments, the power sourceneed not be physically connected to the device.

“Gingivitis” as used herein refers to a non-destructive periodontaldisease that is a response to bacterial biofilms that adhere to thesurface of the teeth. Gingivitis manifests itself in gum tissue throughswollen gums, bright red or purple gums, bleeding gums, receding gums,formation of deep pockets between teeth and gums, loose or shiftingteeth, or bad breath.

“Matrix” or “matrices” as used herein refer to a pattern or patterns,such as those formed by electrodes on a surface, such as a fabric or afiber, or the like. Matrices can be designed to vary the electric fieldor electric current or microcurrent generated. For example, the strengthand shape of the field or current or microcurrent can be altered, or thematrices can be designed to produce an electric field(s) or current ormicrocurrent of a desired strength or shape.

“Oral cavity” as used herein refers to the surfaces inside the mouth,such as hard palate, soft palate, tongue, gingivae, molars, premolars,canine, incisors, frenulum of lower and upper lip, superior and inferiorvestibule, inside of cheek, or any surface within the mouth. The oralcavity is comprised of many surfaces, each with large amounts ofbacteria, and bacterial biofilm.

“Oral cavity device” as used herein refers to a mouth guard, mouth tray,dentures, dental bridge, dental retainer, braces, dental tray, or anytype of device that can be placed within the oral cavity.

“Oral cavity repair” or “oral cavity rejuvenation” as used herein refersto any bacteria, fungus, surgery, bite, cut, abrasion, modification,gingivitis, periodontitis, or the like that is within the oral cavitythat is in need of healing, soothing, restoration, rebuilding, or thelike.

“Periodontitis” as used herein refers to an inflammatory disease thataffects the periodontium, i.e., the tissues that surround and supportthe teeth. Periodontitis can be defined as an inflammation or infectionthat spreads from the gums (gingiva) to the ligaments and bones thatsupport the teeth. Periodontitis can occur by build up of plaque andtartar at the base of the teeth causing swelling traps for bacteria andplaque to form between the gums and the teeth. The term “plaque” can bedefined as a biofilm that develops naturally on teeth, which is formedby colonizing bacteria trying to attach themselves to the tooth's smoothsurface.

“Reduction-oxidation reaction” or “redox reaction” as used herein refersto a reaction involving the transfer of one or more electrons from areducing agent to an oxidizing agent. The term “reducing agent” can bedefined in some embodiments as a reactant in a redox reaction, whichdonates electrons to a reduced species. A “reducing agent” is therebyoxidized in the reaction. The term “oxidizing agent” can be defined insome embodiments as a reactant in a redox reaction, which acceptselectrons from the oxidized species. An “oxidizing agent” is therebyreduced in the reaction. In various embodiments a redox reactionproduced between a first and second reservoir provides a current betweenthe dissimilar reservoirs. The redox reactions can occur spontaneouslywhen a conductive material is brought in proximity to first and seconddissimilar reservoirs such that the conductive material provides amedium for electrical communication and/or ionic communication betweenthe first and second dissimilar reservoirs. In other words, in anembodiment electrical currents can be produced between first and seconddissimilar reservoirs without the use of an external battery or otherpower source (e.g., a direct current (DC) such as a battery or analternating current (AC) power source such as a typical electricoutlet). Accordingly, in various embodiments a system is provided whichis “electrically self contained,” and yet the system can be activated toproduce electrical currents. The term “electrically self contained” canbe defined in some embodiments as being capable of producing electricity(e.g., producing currents) without an external battery or power source.The term “activated” can be defined in some embodiments to refer to theproduction of electric current through the application of a radio signalof a given frequency or through ultrasound or through electromagneticinduction. In other embodiments, a system can be provided which includesan external battery or power source. For example, an AC power source canbe of any wave form, such as a sine wave, a triangular wave, or a squarewave. AC power can also be of any frequency such as for example 50 Hz or60 HZ, or the like. AC power can also be of any voltage, such as forexample 120 volts, or 220 volts, or the like. In embodiments an AC powersource can be electronically modified, such as for example having thevoltage reduced, prior to use.

“Stretchable” as used herein refers to the ability of embodiments thatstretch without losing their structural integrity. That is, embodimentscan stretch to accommodate irregular skin surfaces or surfaces whereinone portion of the surface can move relative to another portion.

LLEC/LLEF Systems, and Devices

In embodiments, devices disclosed herein comprise patterns ofmicro-batteries that create a field between each dot pair. Inembodiments, the unique field is very short, e.g. in the range ofphysiologic electric fields. In embodiments, the direction of theelectric field produced by devices disclosed herein is omnidirectionalover the surface of the wound and more in line with the physiologic.

Embodiments of the LLEC or LLEF system disclosed herein can compriseelectrodes or microcells. Each electrode or microcell can be or includea conductive metal. In embodiments, the electrodes or microcells cancomprise any electrically-conductive material, for example, anelectrically conductive hydrogel, metals, electrolytes, superconductors,semiconductors, plasmas, and nonmetallic conductors such as graphite andconductive polymers. Electrically conductive metals can include silver,copper, gold, aluminum, molybdenum, zinc, lithium, tungsten, brass,carbon, nickel, iron, palladium, platinum, tin, bronze, carbon steel,lead, titanium, stainless steel, mercury, Fe/Cr alloys, and the like.The electrode can be coated or plated with a different metal such asaluminum, gold, platinum or silver.

In certain embodiments, reservoir or electrode geometry can comprisecircles, polygons, lines, zigzags, ovals, stars, or any suitable varietyof shapes. This provides the ability to design/customize surfaceelectric field shapes as well as depth of penetration. For example. Inembodiments it can be desirable to employ an electric field of greaterstrength or depth in an area where skin is thicker to achieve optimaltreatment.

Reservoir or electrode or dot sizes and concentrations can vary, asthese variations can allow for changes in the properties of the electricfield created by embodiments of the invention. Certain embodimentsprovide an electric field at about 1 Volt and then, under normal tissueloads with resistance of 100 k to 300 K ohms, produce a current in therange of 10 microamperes. The electric field strength can be determinedby calculating % the separation distance and applying it in the z-axisover the midpoint between the cell. This indicates the theoreticallocation of the highest strength field line.

Embodiments disclosed herein can comprise patterns of microcells. Thepatterns can be designed to produce an electric field, an electriccurrent, or both, over and through tissue such as human skin. Inembodiments the pattern can be designed to produce a specific size,strength, density, shape, or duration of electric field or electriccurrent. In embodiments reservoir or dot size and separation can bealtered.

In embodiments devices disclosed herein can apply an electric field, anelectric current, or both, wherein the field, current, or both can be ofvarying size, strength, density, shape, or duration in different areasof the embodiment. In embodiments, by micro-sizing the electrodes orreservoirs, the shapes of the electric field, electric current, or bothcan be customized, increasing or decreasing very localized wattdensities and allowing for the design of patterns of electrodes orreservoirs wherein the amount of electric field over a tissue can bedesigned or produced or adjusted based upon feedback from the tissue orupon an algorithm within sensors operably connected to the embodimentand a control module. The electric field, electric current, or both canbe stronger in one zone and weaker in another. The electric field,electric current, or both can change with time and be modulated based ontreatment goals or feedback from the tissue or patient. The controlmodule can monitor and adjust the size, strength, density, shape, orduration of electric field or electric current based on tissueparameters. For example, embodiments disclosed herein can produce andmaintain very localized electrical events. For example, embodimentsdisclosed herein can produce specific values for the electric fieldduration, electric field size, electric field shape, field depth,current, polarity, and/or voltage of the device or system.

A system or device disclosed herein and placed over tissue such as skincan move relative to the tissue. Reducing the amount of motion betweentissue and device can be advantageous to pre-treatment. Slotting orplacing cuts into the device can result in less friction or tension onthe skin. In embodiments, use of an elastic dressing similar to theelasticity of the skin is also possible.

Devices disclosed herein can generate a localized electric field in apattern determined by the distance and physical orientation of the cellsor electrodes. Effective depth of the electric field can bepredetermined by the orientation and distance between the cells orelectrodes.

In embodiments the electric field can be extended, for example throughthe use of a hydrogel. In certain embodiments, for example treatmentmethods, it can be preferable to utilize AC or DC current. For example,embodiments disclosed herein can employ phased array, pulsed, squarewave, sinusoidal, or other wave forms, combinations, or the like.Certain embodiments utilize a controller to produce and control powerproduction and/or distribution to the device.

Embodiments disclosed herein comprise biocompatible electrodes orreservoirs or dots on a surface or substrate, for example a fabric, amouth guard, a mouth tray, a dental retainer, dental bridges, dentures,or the like. Embodiments disclosed herein can be used to treatdisruptions or disrupted spaces in the oral cavity. In embodiments thesurface can be pliable, for example to better follow the contours of anarea to be treated, such as the contour of an individual's gums orteeth. In embodiments the surface can comprise a gauze or mesh orplastic. In embodiments the system comprises a component such as velcro,gecko glue, or, adhesives that are water resistant, or other types ofattachment component to maintain or help maintain its position in oraround the oral cavity device, for example, a mouth guard, mouth tray,dental retainer, dental bridge, dentures, dental tray, or the like. Infurther embodiments the mouth guard, mouth tray, dental retainer, dentalbridge, dentures, dental tray or the like can comprise a conductivematerial, for example a wire to electrically link the device with othercomponents, such as monitoring equipment or a power source. Inembodiments the device can be wirelessly linked to monitoring or datacollection equipment, for example linked via Bluetooth to a cell phonethat collects data from the device. In certain embodiments the devicecan comprise data collection means, such as temperature, pressure, orconductivity data collection. In embodiments the device can be shaped tofit an area of desired use, for example the upper gums, the lower gums,the roof of the oral cavity, the bottom of the oral cavity, or any areawhere gum repair or rejuvenation is desired. For example, in embodimentsthe device can be shaped to fit any type of mouth size or shape thatvaries from subject to subject. In embodiments the device can be shapedto fit an area of desired use, for example primary teeth, permanentteeth, maxillary teeth, mandibular teeth, incisors, canines, premolars,molars or any area where teeth repair and rejuvenation is desired. Inembodiments the device can be a two, three, four, or the like part pastethat when mixed creates a LLEC or LLEF on the subjects teeth and gums.In embodiments the paste can be for example, tooth paste, dental cream,or the like. The paste can react or activate when combined together, orwhen subjected to saliva, hydrogel, or the like. In embodiments thedevice can be a two, three, four, or the like part paste that does notmix together, but is separated by the subjects teeth, gums, or the likeand creates a LLEC or LLEF on the subjects oral cavity. In embodimentsthe paste can be for example, tooth paste, dental cream, or the like.The paste can react or activate when the parts are in close proximitywith each other or touching, and subjected to saliva, hydrogel, or thelike.

Embodiments can include coatings on the surface, such as, for example,over or between the electrodes or cells. Such coatings can include, forexample, silicone, and electrolytic mixture, hypoallergenic agents,drugs, biologics, stem cells, skin substitutes, cosmetic products,combinations, or the like. Drugs suitable for use with embodiments ofthe invention include analgesics, antibiotics, anti-inflammatories, orthe like.

In embodiments the material can include a port to access the interior ofthe material, for example to add fluid, gel, cosmetic products, ahydrating material, or some other material to the dressing. Certainembodiments can comprise a “blister” top that can enclose a materialsuch as an antibacterial. In embodiments the blister top can contain amaterial that is released into or on to the material when the blister ispressed, for example a liquid or cream. For example, embodimentsdisclosed herein can comprise a blister top containing an antibacterialor the like. Embodiments can change color when activated, for examplewhen producing an electric current.

In embodiments the system comprises a component such as elastic tomaintain or help maintain its position. In embodiments the systemcomprises components such as straps to maintain or help maintain itsposition. In certain embodiments the system or device comprises a strapon either end of the long axis, or a strap linking on end of the longaxis to the other. In embodiments that straps can comprise velcro or asimilar fastening system. In embodiments the straps can comprise elasticmaterials. In further embodiments the strap can comprise a conductivematerial, for example a wire to electrically link the device with othercomponents, such as monitoring equipment or a power source. Inembodiments the device can be wirelessly linked to monitoring or datacollection equipment, for example linked via Bluetooth to a cell phoneor computer that collects data from the device. In certain embodimentsthe device can comprise data collection means, such as temperature, pH,pressure, or conductivity data collection means.

In embodiments the system comprises a component such as an adhesive orstraps to maintain or help maintain its position. The adhesive componentcan be covered with a protective layer that is removed to expose theadhesive at the time of use. In embodiments the adhesive can comprise,for example, sealants, such as hypoallergenic sealants, gecko sealants,mussel sealants, waterproof sealants such as epoxies, and the like.Straps can include velcro or similar materials to aid in maintaining theposition of the device.

In embodiments the positioning component can comprise an elastic filmwith an elasticity, for example, similar to that of skin, or greaterthan that of skin, or less than that of skin. In embodiments, the LLECor LLEF system can comprise a laminate where layers of the laminate canbe of varying elasticities. For example, an outer layer may be highlyelastic and an inner layer in-elastic or less elastic. The in-elasticlayer can be made to stretch by placing stress relieving discontinuousregions or slits through the thickness of the material so there is amechanical displacement rather than stress that would break the fabricweave before stretching would occur. In embodiments the slits can extendcompletely through a layer or the system or can be placed whereexpansion is required. In embodiments of the system the slits do notextend all the way through the system or a portion of the system such asthe substrate. In embodiments the discontinuous regions can pass halfwaythrough the long axis of the substrate.

In embodiments the device can be shaped to fit an area of desired use,for example a subject's face, or around a subject's eyes, or around asubject's forehead, a subject's cheeks, a subject's chin, a subject'sback, a subject's chest, a subject's nose, or any area where surgicalpre-treatment is desired. For example, in embodiments the device can beshaped to fit an area where a surgical incision will be made.

Embodiments disclosed herein comprise biocompatible electrodes orreservoirs or dots on a surface or substrate, for example a fabric, afiber, or the like. In embodiments the surface can be pliable, forexample to better follow the contours of an area to be treated, such asthe face or back. In embodiments the surface can comprise a gauze ormesh or plastic. Suitable types of pliable surfaces for use inembodiments disclosed herein can be absorbent or non-absorbent textiles,low-adhesives, vapor permeable films, hydrocolloids, hydrogels,alginates, foams, foam-based materials, cellulose-based materialsincluding Kettenbach fibers, hollow tubes, fibrous materials, such asthose impregnated with anhydrous/hygroscopic materials, beads and thelike, or any suitable material as known in the art. In embodiments thepliable material can form, for example, a mask, such as that worn on theface, an eye patch, a shirt or a portion thereof, for example an elasticor compression shirt, or a portion thereof, a wrapping, towel, cloth,fabric, or the like. Multi layer embodiments can include, for example, askin-contacting layer, a hydration layer, and a hydration containmentlayer.

An electroceutical fabric can be produced by weaving fibers whereinsections of the fibers are coated or treated with materials capable ofproducing electricity and forming a battery in the presence of anelectrolyte.

In embodiments, the fabric can be woven of at least two types of fibers;fibers comprising sections treated or coated with a substance capable offorming a positive electrode; and fibers comprising sections treated orcoated with a substance capable of forming a negative electrode. Thefabric can further comprise fibers that do not form an electrode. Longlengths of fibers can be woven together to form fabrics. For example,the fibers can be woven together to form a regular pattern of positiveand negative electrodes.

In an embodiment, fibers are produced with discrete sections coated atregular intervals with silver (as seen in FIG. 14 ; discrete sections140). Fibers are produced with discrete sections coated at regularintervals with zinc (as seen in FIG. 14 ; discrete sections 145). Thefibers can be coated with, for example, a printer, such as a laser orink-jet printer. In embodiments the fibers can be spray-coated. Inembodiments the fibers can be dip-coated.

In embodiments, continuous fibers can be produced with sections of thefiber coated with zinc. In embodiments, continuous fibers can beproduced with sections of the fiber coated with silver.

Embodiments can comprise a moisture-sensitive component that changescolor when the device is activated and producing an electric current.

In embodiments, the fibers can be woven into a pattern (as seen in FIG.2 ) to produce fabric. In embodiments, the fabric can be hydrated, forexample with an electrolyte, to produce a voltage between theelectrodes.

Embodiments disclosed herein or produced by the methods disclosed hereincan comprise “anchor” regions or “arms” or straps to affix the systemsecurely. The anchor regions or arms can anchor the LLEC system. Forexample, a LLEC system can be secured to an area proximal to a joint orirregular skin surface, and anchor regions of the system can extend toareas of minimal stress or movement to securely affix the system.Further, the LLEC system can reduce stress on an area, for example by“countering” the physical stress caused by movement.

In embodiments the LLEC or LLEF system can comprise additionaltherapeutic materials.

In embodiments, the LLEC or LLEF system can comprise instructions ordirections on how to place the system to maximize its performance.

A LLEC or LLEF system disclosed herein can comprise “anchor” regions or“arms” or straps to affix the system securely. The anchor regions orarms can anchor the LLEC or LLEF system. For example, a LLEC or LLEFsystem can be secured to an area proximal to a joint or irregular skinsurface, and anchor regions of the system can extend to areas of minimalstress or movement to securely affix the system. Further, the LLECsystem can reduce stress on an area, for example by “countering” thephysical stress caused by movement.

In embodiments the LLEC or LLEF system can comprise additional materialsto aid in treatment.

Embodiments can include devices in the form of a gel, such as, forexample, a one-or two-component gel that is mixed on use. Embodimentscan include devices in the form of a spray, for example, a one- ortwo-component spray that is mixed on use.

In embodiments, the LLEC or LLEF system can comprise instructions ordirections on how to place the system to maximize its performance.Embodiments include a kit comprising an LLEC or LLEF system anddirections for its use.

In certain embodiments dissimilar metals can be used to create anelectric field with a desired voltage. In certain embodiments thepattern of reservoirs can control the watt density and shape of theelectric field.

Certain embodiments can utilize a power source to create the electriccurrent, such as a battery or a micro-battery. The power source can beany energy source capable of generating a current in the LLEC system andcan include, for example, AC power, DC power, radio frequencies (RF)such as pulsed RF, induction, ultrasound, and the like.

Dissimilar metals used to make a LLEC or LLEF system disclosed hereincan be silver and zinc, and the electrolytic solution can include sodiumchloride in water. In certain embodiments the electrodes are appliedonto a non-conductive surface to create a pattern, most preferably anarray or multi-array of voltaic cells that do not spontaneously reactuntil they contact an electrolytic solution. Sections of thisdescription use the terms “printing” with “ink,” but it is to beunderstood that the patterns may also be “painted” with “paints.” Theuse of any suitable means for applying a conductive material iscontemplated. In embodiments “ink” or “paint” can comprise any solutionsuitable for forming an electrode on a surface such as a conductivematerial including a conductive metal solution. In embodiments“printing” or “painted” can comprise any method of applying a solutionto a material upon which a matrix is desired.

A preferred material to use in combination with silver to create thevoltaic cells or reservoirs of disclosed embodiments is zinc. Zinc hasbeen well-described for its uses in prevention of infection in suchtopical antibacterial agents as Bacitracin zinc, a zinc salt ofBacitracin. Zinc is a divalent cation with antibacterial properties ofits own.

Turning to the figures, in FIG. 1 , the dissimilar first electrode 6 andsecond electrode 10 are applied onto a desired primary surface 2 of anarticle 4, for example a fabric. In one embodiment a primary surface isa surface of a LLEC or LLEF system that comes into direct contact withan area to be treated such as a skin surface.

In various embodiments the difference of the standard potentials of theelectrodes or dots or reservoirs can be in a range from about 0.05 V toapproximately about 5.0 V. For example, the standard potential can beabout 0.05 V, about 0.06 V, about 0.07 V, about 0.08 V, about 0.09 V,about 0.1 V, about 0.2 V, about 0.3 V, about 0.4 V, about 0.5 V, about0.6 V, about 0.7 V, about 0.8 V, about 0.9 V, about 1.0 V, about 1.1 V,about 1.2 V, about 1.3 V, about 1.4 V, about 1.5 V, about 1.6 V, about1.7 V, about 1.8 V, about 1.9 V, about 2.0 V, about 2.1 V, about 2.2 V,about 2.3 V, about 2.4 V, about 2.5 V, about 2.6 V, about 2.7 V, 2.8 V,about 2.9 V, about 3.0 V, about 3.1 V, about 3.2 V, about 3.3 V, about3.4 V, about 3.5 V, about 3.6 V, about 3.7 V, about 3.8 V, about 3.9 V,about 4.0 V, about 4.1 V, about 4.2 V, about 4.3 V, 4.4 V, 4.5 V, about4.6 V, about 4.7 V, 4.8 V, about 4.9 V, about 5.0 V, about 5.1 V, about5.2 V, about 5.3 V, about 5.4 V, about 5.5 V, about 5.6 V, about 5.7 V,about 5.8 V, about 5.9 V, about 6.0 V, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelmicro-current of between for example about 1 and about 200micro-amperes, between about 10 and about 190 micro-amperes, betweenabout 20 and about 180 micro-amperes, between about 30 and about 170micro-amperes, between about 40 and about 160 micro-amperes, betweenabout 50 and about 150 micro-amperes, between about 60 and about 140micro-amperes, between about 70 and about 130 micro-amperes, betweenabout 80 and about 120 micro-amperes, between about 90 and about 100micro-amperes, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelmicro-current of between for example about 1 and about 400micro-amperes, between about 20 and about 380 micro-amperes, betweenabout 400 and about 360 micro-amperes, between about 60 and about 340micro-amperes, between about 80 and about 320 micro-amperes, betweenabout 100 and about 3000 micro-amperes, between about 120 and about 280micro-amperes, between about 140 and about 260 micro-amperes, betweenabout 160 and about 240 micro-amperes, between about 180 and about 220micro-amperes, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelmicro-current about 10 micro-amperes, about 20 micro-amperes, about 30micro-amperes, about 40 micro-amperes, about 50 micro-amperes, about 60micro-amperes, about 70 micro-amperes, about 80 micro-amperes, about 90micro-amperes, about 100 micro-amperes, about 110 micro-amperes, about120 micro-amperes, about 130 micro-amperes, about 140 micro-amperes,about 150 micro-amperes, about 160 micro-amperes, about 170micro-amperes, about 180 micro-amperes, about 190 micro-amperes, about200 micro-amperes, about 210 micro-amperes, about 220 micro-amperes,about 240 micro-amperes, about 260 micro-amperes, about 280micro-amperes, about 300 micro-amperes, about 320 micro-amperes, about340 micro-amperes, about 360 micro-amperes, about 380 micro-amperes,about 400 micro-amperes, or the like.

In embodiments, the disclosed LLEC systems can produce a low levelmicro-current of not more than 10 micro-amperes, or not more than about20 micro-amperes, not more than about 30 micro-amperes, not more thanabout 40 micro-amperes, not more than about 50 micro-amperes, not morethan about 60 micro-amperes, not more than about 70 micro-amperes, notmore than about 80 micro-amperes, not more than about 90 micro-amperes,not more than about 100 micro-amperes, not more than about 110micro-amperes, not more than about 120 micro-amperes, not more thanabout 130 micro-amperes, not more than about 140 micro-amperes, not morethan about 150 micro-amperes, not more than about 160 micro-amperes, notmore than about 170 micro-amperes, not more than about 180micro-amperes, not more than about 190 micro-amperes, not more thanabout 200 micro-amperes, not more than about 210 micro-amperes, not morethan about 220 micro-amperes, not more than about 230 micro-amperes, notmore than about 240 micro-amperes, not more than about 250micro-amperes, not more than about 260 micro-amperes, not more thanabout 270 micro-amperes, not more than about 280 micro-amperes, not morethan about 290 micro-amperes, not more than about 300 micro-amperes, notmore than about 310 micro-amperes, not more than about 320micro-amperes, not more than about 340 micro-amperes, not more thanabout 360 micro-amperes, not more than about 380 micro-amperes, not morethan about 400 micro-amperes, not more than about 420 micro-amperes, notmore than about 440 micro-amperes, not more than about 460micro-amperes, not more than about 480 micro-amperes, or the like.

In embodiments, LLEC systems disclosed herein can produce a low levelmicro-current of not less than 10 micro-amperes, not less than 20micro-amperes, not less than 30 micro-amperes, not less than 40micro-amperes, not less than 50 micro-amperes, not less than 60micro-amperes, not less than 70 micro-amperes, not less than 80micro-amperes, not less than 90 micro-amperes, not less than 100micro-amperes, not less than 110 micro-amperes, not less than 120micro-amperes, not less than 130 micro-amperes, not less than 140micro-amperes, not less than 150 micro-amperes, not less than 160micro-amperes, not less than 170 micro-amperes, not less than 180micro-amperes, not less than 190 micro-amperes, not less than 200micro-amperes, not less than 210 micro-amperes, not less than 220micro-amperes, not less than 230 micro-amperes, not less than 240micro-amperes, not less than 250 micro-amperes, not less than 260micro-amperes, not less than 270 micro-amperes, not less than 280micro-amperes, not less than 290 micro-amperes, not less than 300micro-amperes, not less than 310 micro-amperes, not less than 320micro-amperes, not less than 330 micro-amperes, not less than 340micro-amperes, not less than 350 micro-amperes, not less than 360micro-amperes, not less than 370 micro-amperes, not less than 380micro-amperes, not less than 390 micro-amperes, not less than 400micro-amperes, or the like.

The applied electrodes or reservoirs or dots can adhere or bond to theprimary surface 2 because a biocompatible binder is mixed, inembodiments into separate mixtures, with each of the dissimilar metalsthat will create the pattern of voltaic cells, in embodiments. Most inksare simply a carrier, and a binder mixed with pigment. Similarly,conductive metal solutions can be a binder mixed with a conductiveelement. The resulting conductive metal solutions can be used with anapplication method such as screen printing to apply the electrodes tothe primary surface in predetermined patterns. Once the conductive metalsolutions dry and/or cure, the patterns of spaced electrodes cansubstantially maintain their relative position, even on a flexiblematerial such as that used for a LLEC or LLEF system. To make a limitednumber of the systems of an embodiment disclosed herein, the conductivemetal solutions can be hand applied onto a common adhesive bandage sothat there is an array of alternating electrodes that are spaced about amillimeter apart on the primary surface of the bandage. The solution canbe allowed to dry before being applied to a surface so that theconductive materials do not mix, which could interrupt the array andcause direct reactions that will release the elements.

In certain embodiments that utilize a poly-cellulose binder, the binderitself can have an beneficial effect such as reducing the localconcentration of matrix metallo-proteases through an iontophoreticprocess that drives the cellulose into the surrounding tissue. Thisprocess can be used to electronically drive other components such asdrugs into the surrounding tissue.

The binder can include any biocompatible liquid material that can bemixed with a conductive element (preferably metallic crystals of silveror zinc) to create a conductive solution which can be applied as a thincoating to a surface. One suitable binder is a solvent reduciblepolymer, such as the polyacrylic non-toxic silk-screen ink manufacturedby COLORCON® Inc., a division of Berwind Pharmaceutical Services, Inc.(see COLORCON® NO-TOX® product line, part number NT28). In an embodimentthe binder is mixed with high purity (at least 99.999%) metallic silvercrystals to make the silver conductive solution. The silver crystals,which can be made by grinding silver into a powder, are preferablysmaller than 100 microns in size or about as fine as flour. In anembodiment, the size of the crystals is about 325 mesh, which istypically about 40 microns in size or a little smaller. The binder isseparately mixed with high purity (at least 99.99%, in an embodiment)metallic zinc powder which has also preferably been sifted throughstandard 325 mesh screen, to make the zinc conductive solution. Forbetter quality control and more consistent results, most of the crystalsused should be larger than 325 mesh and smaller than 200 mesh. Forexample the crystals used should be between 200 mesh and 325 mesh, orbetween 210 mesh and 310 mesh, between 220 mesh and 300 mesh, between230 mesh and 290 mesh, between 240 mesh and 280 mesh, between 250 meshand 270 mesh, between 255 mesh and 265 mesh, or the like.

Other powders of metal can be used to make other conductive metalsolutions in the same way as described in other embodiments.

The size of the metal crystals, the availability of the surface to theconductive fluid and the ratio of metal to binder affects the releaserate of the metal from the mixture. When COLORCON® polyacrylic ink isused as the binder, about 10 to 40 percent of the mixture should bemetal for a longer term bandage (for example, one that stays on forabout 10 days). For example, for a longer term LLEC or LLEF system thepercent of the mixture that should be metal can be 8 percent, or 10percent, 12 percent, 14 percent, 16 percent, 18 percent, 20 percent, 22percent, 24 percent, 26 percent, 28 percent, 30 percent, 32 percent, 34percent, 36 percent, 38 percent, 40 percent, 42 percent, 44 percent, 46percent, 48 percent, 50 percent, or the like.

If the same binder is used, but the percentage of the mixture that ismetal is increased to 60 percent or higher, then the release rate willbe much faster and a typical system will only be effective for a fewdays. For example, for a shorter term device, the percent of the mixturethat should be metal can be 40 percent, or 42 percent, 44 percent, 46percent, 48 percent, 50 percent, 52 percent, 54 percent, 56 percent, 58percent, 60 percent, 62 percent, 64 percent, 66 percent, 68 percent, 70percent, 72 percent, 74 percent, 76 percent, 78 percent, 80 percent, 82percent, 84 percent, 86 percent, 88 percent, 90 percent, or the like.

For LLEC or LLEF systems comprising a pliable substrate it can bedesired to decrease the percentage of metal down to 5 percent or less,or to use a binder that causes the crystals to be more deeply embedded,so that the primary surface will be antimicrobial for a very long periodof time and will not wear prematurely. Other binders can dissolve orotherwise break down faster or slower than a polyacrylic ink, soadjustments can be made to achieve the desired rate of spontaneousreactions from the voltaic cells.

To maximize the number of voltaic cells, in various embodiments, apattern of alternating silver masses or electrodes or reservoirs andzinc masses or electrodes or reservoirs can create an array ofelectrical currents across the primary surface. A basic pattern, shownin FIG. 1 , has each mass of silver equally spaced from four masses ofzinc, and has each mass of zinc equally spaced from four masses ofsilver, according to an embodiment. The first electrode 6 is separatedfrom the second electrode 10 by a spacing 8. The designs of firstelectrode 6 and second electrode 10 are simply round dots, and in anembodiment, are repeated. Numerous repetitions 12 of the designs resultin a pattern. For an exemplary device comprising silver and zinc, eachsilver design preferably has about twice as much mass as each zincdesign, in an embodiment. For the pattern in FIG. 1 , the silver designsare most preferably about a millimeter from each of the closest fourzinc designs, and vice-versa. The resulting pattern of dissimilar metalmasses defines an array of voltaic cells when introduced to anelectrolytic solution. Further disclosure relating to methods ofproducing micro-arrays can be found in U.S. Pat. No. 7,813,806 entitledCURRENT PRODUCING SURFACE FOR TREATING BIOLOGIC TISSUE issued Oct. 12,2010, which is incorporated by reference in its entirety.

A dot pattern of masses like the alternating round dots of FIG. 1 can bepreferred when applying conductive material onto a flexible material,such as those used for a facial or eye mask, or an article of clothingsuch as a shirt or shorts, as the dots won't significantly affect theflexibility of the material. To maximize the density of electricalcurrent over a primary surface the pattern of FIG. 2 can be used. Thefirst electrode 6 in FIG. 2 is a large hexagonally shaped dot, and thesecond electrode 10 is a pair of smaller hexagonally shaped dots thatare spaced from each other. The spacing 8 that is between the firstelectrode 6 and the second electrode 10 maintains a relativelyconsistent distance between adjacent sides of the designs. Numerousrepetitions 12 of the designs result in a pattern 14 that can bedescribed as at least one of the first design being surrounded by sixhexagonally shaped dots of the second design.

FIGS. 3 and 4 show how the pattern of FIG. 2 can be used to make anembodiment disclosed herein. The pattern shown in detail in FIG. 2 isapplied to the primary surface 2 of an embodiment. The back 20 of theprinted material is fixed to a substrate layer 22. This layer isadhesively fixed to a pliable layer 16.

FIG. 5 shows an additional feature, which can be added between designs,that can initiate the flow of current in a poor electrolytic solution. Afine line 24 is printed using one of the conductive metal solutionsalong a current path of each voltaic cell. The fine line will initiallyhave a direct reaction but will be depleted until the distance betweenthe electrodes increases to where maximum voltage is realized. Theinitial current produced is intended to help control edema so that theLLEC system will be effective. If the electrolytic solution is highlyconductive when the system is initially applied the fine line can bequickly depleted and the device will function as though the fine linehad never existed.

FIGS. 6 and 7 show alternative patterns that use at least one linedesign. The first electrode 6 of FIG. 6 is a round dot similar to thefirst design used in FIG. 1 . The second electrode 10 of FIG. 6 is aline. When the designs are repeated, they define a pattern of parallellines that are separated by numerous spaced dots. FIG. 7 uses only linedesigns. The first electrode 6 can be thicker or wider than the secondelectrode 10 if the oxidation-reduction reaction requires more metalfrom the first conductive element (mixed into the first design'sconductive metal solution) than the second conductive element (mixedinto the second design's conductive metal solution). The lines can bedashed. Another pattern can be silver grid lines that have zinc massesin the center of each of the cells of the grid. The pattern can beletters printed from alternating conductive materials so that a messagecan be printed onto the primary surface-perhaps a brand name oridentifying information such as patient blood type.

Because the spontaneous oxidation-reduction reaction of silver and zincuses a ratio of approximately two silver to one zinc, the silver designcan contain about twice as much mass as the zinc design in anembodiment. At a spacing of about 1 mm between the closest dissimilarmetals (closest edge to closest edge) each voltaic cell that contacts aconductive fluid such as a cosmetic cream can create approximately 1volt of potential that will penetrate substantially through the dermisand epidermis. Closer spacing of the dots can decrease the resistance,providing less potential, and the current will not penetrate as deeply.If the spacing falls below about one tenth of a millimeter a benefit ofthe spontaneous reaction is that which is also present with a directreaction; silver can be electrically driven into the skin. Therefore,spacing between the closest conductive materials can be, for example,0.1 mm, or 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9mm, 1 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4 mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8mm, 1.9 mm, 2 mm, 2.1 mm, 2.2 mm, 2.3 mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7mm, 2.8 mm, 2.9 mm, 3 mm, 3.1 mm, 3.2 mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6mm, 3.7 mm, 3.8 mm, 3.9 mm, 4 mm, 4.1 mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5 mm, 5.1 mm, 5.2 mm, 5.3 mm, 5.4mm, 5.5 mm, 5.6 mm, 5.7 mm, 5.8 mm, 5.9 mm, 6 mm, or the like.

In certain embodiments the spacing between the closest conductivematerials can be not more than 0.1 mm, or not more than 0.2 mm, not morethan 0.3 mm, not more than 0.4 mm, not more than 0.5 mm, not more than0.6 mm, not more than 0.7 mm, not more than 0.8 mm, not more than 0.9mm, not more than 1 mm, not more than 1.1 mm, not more than 1.2 mm, notmore than 1.3 mm, not more than 1.4 mm, not more than 1.5 mm, not morethan 1.6 mm, not more than 1.7 mm, not more than 1.8 mm, not more than1.9 mm, not more than 2 mm, not more than 2.1 mm, not more than 2.2 mm,not more than 2.3 mm, not more than 2.4 mm, not more than 2.5 mm, notmore than 2.6 mm, not more than 2.7 mm, not more than 2.8 mm, not morethan 2.9 mm, not more than 3 mm, not more than 3.1 mm, not more than 3.2mm, not more than 3.3 mm, not more than 3.4 mm, not more than 3.5 mm,not more than 3.6 mm, not more than 3.7 mm, not more than 3.8 mm, notmore than 3.9 mm, not more than 4 mm, not more than 4.1 mm, not morethan 4.2 mm, not more than 4.3 mm, not more than 4.4 mm, not more than4.5 mm, not more than 4.6 mm, not more than 4.7 mm, not more than 4.8mm, not more than 4.9 mm, not more than 5 mm, not more than 5.1 mm, notmore than 5.2 mm, not more than 5.3 mm, not more than 5.4 mm, not morethan 5.5 mm, not more than 5.6 mm, not more than 5.7 mm, not more than5.8 mm, not more than 5.9 mm, not more than 6 mm, or the like.

In certain embodiments spacing between the closest conductive materialscan be not less than 0.1 mm, or not less than 0.2 mm, not less than 0.3mm, not less than 0.4 mm, not less than 0.5 mm, not less than 0.6 mm,not less than 0.7 mm, not less than 0.8 mm, not less than 0.9 mm, notless than 1 mm, not less than 1.1 mm, not less than 1.2 mm, not lessthan 1.3 mm, not less than 1.4 mm, not less than 1.5 mm, not less than1.6 mm, not less than 1.7 mm, not less than 1.8 mm, not less than 1.9mm, not less than 2 mm, not less than 2.1 mm, not less than 2.2 mm, notless than 2.3 mm, not less than 2.4 mm, not less than 2.5 mm, not lessthan 2.6 mm, not less than 2.7 mm, not less than 2.8 mm, not less than2.9 mm, not less than 3 mm, not less than 3.1 mm, not less than 3.2 mm,not less than 3.3 mm, not less than 3.4 mm, not less than 3.5 mm, notless than 3.6 mm, not less than 3.7 mm, not less than 3.8 mm, not lessthan 3.9 mm, not less than 4 mm, not less than 4.1 mm, not less than 4.2mm, not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm,not less than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, notless than 4.9 mm, not less than 5 mm, not less than 5.1 mm, not lessthan 5.2 mm, not less than 5.3 mm, not less than 5.4 mm, not less than5.5 mm, not less than 5.6 mm, not less than 5.7 mm, not less than 5.8mm, not less than 5.9 mm, not less than 6 mm, or the like.

Disclosures of the present specification include LLEC or LLEF systemscomprising a primary surface of a pliable material wherein the pliablematerial is adapted to be applied to an area of tissue such as the faceof a subject; a first electrode design formed from a first conductiveliquid that includes a mixture of a polymer and a first element, thefirst conductive liquid being applied into a position of contact withthe primary surface, the first element including a metal species, andthe first electrode design including at least one dot or reservoir,wherein selective ones of the at least one dot or reservoir haveapproximately a 1.5 mm +/− 1-1 mm mean diameter; a second electrodedesign formed from a second conductive liquid that includes a mixture ofa polymer and a second element, the second element including a differentmetal species than the first element, the second conductive liquid beingprinted into a position of contact with the primary surface, and thesecond electrode design including at least one other dot or reservoir,wherein selective ones of the at least one other dot or reservoir haveapproximately a 2.5 mm +/− 1-2 mm mean diameter; a spacing on theprimary surface that is between the first electrode design and thesecond electrode design such that the first electrode design does notphysically contact the second electrode design, wherein the spacing isapproximately 1.5 mm +/− 1-1 mm, and at least one repetition of thefirst electrode design and the second electrode design, the at least onerepetition of the first electrode design being substantially adjacentthe second electrode design, wherein the at least one repetition of thefirst electrode design and the second electrode design, in conjunctionwith the spacing between the first electrode design and the secondelectrode design, defines at least one pattern of at least one voltaiccell for spontaneously generating at least one electrical current whenintroduced to an electrolytic solution. Therefore, electrodes, dots orreservoirs can have a mean diameter of 0.2 mm, or 0.3 mm, 0.4 mm, 0.5mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.1 mm, 1.2 mm, 1.3 mm, 1.4mm, 1.5 mm, 1.6 mm, 1.7 mm, 1.8 mm, 1.9 mm, 2.0 mm, 2.1 mm, 2.2 mm, 2.3mm, 2.4 mm, 2.5 mm, 2.6 mm, 2.7 mm, 2.8 mm, 2.9 mm, 3.0 mm, 3.1 mm, 3.2mm, 3.3 mm, 3.4 mm, 3.5 mm, 3.6 mm, 3.7 mm, 3.8 mm, 3.9 mm, 4.0 mm, 4.1mm, 4.2 mm, 4.3 mm, 4.4 mm, 4.5 mm, 4.6 mm, 4.7 mm, 4.8 mm, 4.9 mm, 5.0mm, or the like.

In further embodiments, electrodes, dots or reservoirs can have a meandiameter of not less than 0.2 mm, or not less than 0.3 mm, not less than0.4 mm, not less than 0.5 mm, not less than 0.6 mm, not less than 0.7mm, not less than 0.8 mm, not less than 0.9 mm, not less than 1.0 mm,not less than 1.1 mm, not less than 1.2 mm, not less than 1.3 mm, notless than 1.4 mm, not less than 1.5 mm, not less than 1.6 mm, not lessthan 1.7 mm, not less than 1.8 mm, not less than 1.9 mm, not less than2.0 mm, not less than 2.1 mm, not less than 2.2 mm, not less than 2.3mm, not less than 2.4 mm, not less than 2.5 mm, not less than 2.6 mm,not less than 2.7 mm, not less than 2.8 mm, not less than 2.9 mm, notless than 3.0 mm, not less than 3.1 mm, not less than 3.2 mm, not lessthan 3.3 mm, not less than 3.4 mm, not less than 3.5 mm, not less than3.6 mm, not less than 3.7 mm, not less than 3.8 mm, not less than 3.9mm, not less than 4.0 mm, not less than 4.1 mm, not less than 4.2 mm,not less than 4.3 mm, not less than 4.4 mm, not less than 4.5 mm, notless than 4.6 mm, not less than 4.7 mm, not less than 4.8 mm, not lessthan 4.9 mm, not less than 5.0 mm, or the like.

In further embodiments, electrodes, dots or reservoirs can have a meandiameter of not more than 0.2 mm, or not more than 0.3 mm, not more than0.4 mm, not more than 0.5 mm, not more than 0.6 mm, not more than 0.7mm, not more than 0.8 mm, not more than 0.9 mm, not more than 1.0 mm,not more than 1.1 mm, not more than 1.2 mm, not more than 1.3 mm, notmore than 1.4 mm, not more than 1.5 mm, not more than 1.6 mm, not morethan 1.7 mm, not more than 1.8 mm, not more than 1.9 mm, not more than2.0 mm, not more than 2.1 mm, not more than 2.2 mm, not more than 2.3mm, not more than 2.4 mm, not more than 2.5 mm, not more than 2.6 mm,not more than 2.7 mm, not more than 2.8 mm, not more than 2.9 mm, notmore than 3.0 mm, not more than 3.1 mm, not more than 3.2 mm, not morethan 3.3 mm, not more than 3.4 mm, not more than 3.5 mm, not more than3.6 mm, not more than 3.7 mm, not more than 3.8 mm, not more than 3.9mm, not more than 4.0 mm, not more than 4.1 mm, not more than 4.2 mm,not more than 4.3 mm, not more than 4.4 mm, not more than 4.5 mm, notmore than 4.6 mm, not more than 4.7 mm, not more than 4.8 mm, not morethan 4.9 mm, not more than 5.0 mm, or the like.

The material concentrations or quantities within and/or the relativesizes (e.g., dimensions or surface area) of the first and secondreservoirs can be selected deliberately to achieve variouscharacteristics of the systems' behavior. For example, the quantities ofmaterial within a first and second reservoir can be selected to providean apparatus having an operational behavior that depletes atapproximately a desired rate and/or that “dies” after an approximateperiod of time after activation. In an embodiment the one or more firstreservoirs and the one or more second reservoirs are configured tosustain one or more currents for an approximate pre-determined period oftime, after activation. It is to be understood that the amount of timethat currents are sustained can depend on external conditions andfactors (e.g., the quantity and type of activation material), andcurrents can occur intermittently depending on the presence or absenceof activation material. Further disclosure relating to producingreservoirs that are configured to sustain one or more currents for anapproximate pre-determined period of time can be found in U.S. Pat. No.7,904,147 entitled SUBSTANTIALLY PLANAR ARTICLE AND METHODS OFMANUFACTURE issued Mar. 8, 2011, which is incorporated by referenceherein in its entirety.

In various embodiments the difference of the standard potentials of thefirst and second reservoirs can be in a range from 0.05 V toapproximately 5.0 V. For example, the standard potential can be 0.05 V,or 0.06 V, 0.07 V, 0.08 V, 0.09 V, 0.1 V, 0.2 V, 0.3 V, 0.4 V, 0.5 V,0.6 V, 0.7 V, 0.8 V, 0.9 V, 1.0 V, 1.1 V, 1.2 V, 1.3 V, 1.4 V, 1.5 V,1.6 V, 1.7 V, 1.8 V, 1.9 V, 2.0 V, 2.1 V, 2.2 V, 2.3 V, 2.4 V, 2.5 V,2.6 V, 2.7 V, 2.8 V, 2.9 V, 3.0 V, 3.1 V, 3.2 V, 3.3 V, 3.4 V, 3.5 V,3.6 V, 3.7 V, 3.8 V, 3.9 V, 4.0 V, 4.1 V, 4.2 V, 4.3 V, 4.4 V, 4.5 V,4.6 V, 4.7 V, 4.8 V, 4.9 V, 5.0 V, or the like.

In a particular embodiment the difference of the standard potentials ofthe first and second reservoirs can be at least 0.05 V, or at least 0.06V, at least 0.07 V, at least 0.08 V, at least 0.09 V, at least 0.1 V, atleast 0.2 V, at least 0.3 V, at least 0.4 V, at least 0.5 V, at least0.6 V, at least 0.7 V, at least 0.8 V, at least 0.9 V, at least 1.0 V,at least 1.1 V, at least 1.2 V, at least 1.3 V, at least 1.4 V, at least1.5 V, at least 1.6 V, at least 1.7 V, at least 1.8 V, at least 1.9 V,at least 2.0 V, at least 2.1 V, at least 2.2 V, at least 2.3 V, at least2.4 V, at least 2.5 V, at least 2.6 V, at least 2.7 V, at least 2.8 V,at least 2.9 V, at least 3.0 V, at least 3.1 V, at least 3.2 V, at least3.3 V, at least 3.4 V, at least 3.5 V, at least 3.6 V, at least 3.7 V,at least 3.8 V, at least 3.9 V, at least 4.0 V, at least 4.1 V, at least4.2 V, at least 4.3 V, at least 4.4 V, at least 4.5 V, at least 4.6 V,at least 4.7 V, at least 4.8 V, at least 4.9 V, at least 5.0 V, or thelike.

In a particular embodiment, the difference of the standard potentials ofthe first and second reservoirs can be not more than 0.05 V, or not morethan 0.06 V, not more than 0.07 V, not more than 0.08 V, not more than0.09 V, not more than 0.1 V, not more than 0.2 V, not more than 0.3 V,not more than 0.4 V, not more than 0.5 V, not more than 0.6 V, not morethan 0.7 V, not more than 0.8 V, not more than 0.9 V, not more than 1.0V, not more than 1.1 V, not more than 1.2 V, not more than 1.3 V, notmore than 1.4 V, not more than 1.5 V, not more than 1.6 V, not more than1.7 V, not more than 1.8 V, not more than 1.9 V, not more than 2.0 V,not more than 2.1 V, not more than 2.2 V, not more than 2.3 V, not morethan 2.4 V, not more than 2.5 V, not more than 2.6 V, not more than 2.7V, not more than 2.8 V, not more than 2.9 V, not more than 3.0 V, notmore than 3.1 V, not more than 3.2 V, not more than 3.3 V, not more than3.4 V, not more than 3.5 V, not more than 3.6 V, not more than 3.7 V,not more than 3.8 V, not more than 3.9 V, not more than 4.0 V, not morethan 4.1 V, not more than 4.2 V, not more than 4.3 V, not more than 4.4V, not more than 4.5 V, not more than 4.6 V, not more than 4.7 V, notmore than 4.8 V, not more than 4.9 V, not more than 5.0 V, or the like.In embodiments that include very small reservoirs (e.g., on thenanometer scale), the difference of the standard potentials can besubstantially less or more. The electrons that pass between the firstreservoir and the second reservoir can be generated as a result of thedifference of the standard potentials. Further disclosure relating tostandard potentials can be found in U.S. Pat. No. 8,224,439 entitledBATTERIES AND METHODS OF MANUFACTURE AND USE issued Jul. 17, 2012, whichis incorporated be reference herein in its entirety.

The voltage present at the site of surgical pre-treatment is typicallyin the range of millivolts but disclosed embodiments can introduce amuch higher voltage, for example near 1 volt when using the 1 mm spacingof dissimilar metals already described. The higher voltage is believedto drive the current deeper into the treatment area. In this way thecurrent not only can drive silver and zinc into the treatment if desiredfor treatment, but the current can also provide a stimulatory current sothat the entire surface area can be treated. The higher voltage may alsoincrease antimicrobial effect bacteria and preventing biofilms. Theelectric field can also have beneficial effects on cell migration, ATPproduction, and angiogenesis.

Embodiments disclosed herein relating to surgical pre-treatment can alsocomprise selecting a patient or tissue in need of, or that could benefitby, surgical pre-treatment.

While various embodiments have been shown and described, it will berealized that alterations and modifications can be made thereto withoutdeparting from the scope of the following claims. It is expected thatother methods of applying the conductive material can be substituted asappropriate. Also, there are numerous shapes, sizes and patterns ofvoltaic cells that have not been described but it is expected that thisdisclosure will enable those skilled in the art to incorporate their owndesigns which will then be applied to a surface to create voltaic cellswhich will become active when brought into contact with an electrolyticsolution.

Certain embodiments include LLEC or LLEF systems comprising embodimentsdesigned to be used on irregular, non-planar, or “stretching” surfaces.Embodiments disclosed herein can be used with numerous irregularsurfaces of the body, including the face, the shoulder, the elbow, thewrist, the finger joints, the hip, the knee, the ankle, the toe joints,etc. Additional embodiments disclosed herein can be used in areas wheretissue is prone to movement, for example the eyelid, the ear, the lips,the nose, the shoulders, the back, etc.

In certain embodiments, the substrate can be shaped to fit a particularregion of the body. As shown in FIG. 9 , a mask-shaped substrate 94 canbe used for the surgical pre-treatment around the face and forehead.Embodiments can also include means for securing the mask 94 to theuser's head 36. In an embodiment the portion of the mask or substratethat is to contact the skin comprises a multi-array matrix ofbiocompatible microcells. In certain embodiments a fluid or cream suchas a conductive antibacterial fluid or cream can be applied between themulti-array matrix of biocompatible microcells and the skin.

Similarly, FIG. 10 depicts an embodiment designed to treat the backprior to surgery. Water impermeable barrier 41 prevents or minimizesliquid from escaping from the device. Hydration material 42 is between41 and a multi-array matrix of biocompatible microcells layer 43. Thehydration material 42 moistens layer 43. Adhesive velcro (or othersuitable adhesion device) areas 44 affix layers 42 and 43 to layer 41.Edging material 45 is soft and flexible and protects the user fromdiscomfort. Shoulder straps 46 are made of a soft, flexible material andensure a secure fit on the patient. Soft, flexible waist strap 47secures the lower portion of the embodiment to the patient.

Various apparatus embodiments which can be referred to as “medicalbatteries” are described herein. Further disclosure relating to thistechnology can be found in U.S. Pat. No. 7,672,719 entitled BATTERIESAND METHODS OF MANUFACTURE AND USE issued Mar. 2, 2010, which isincorporated herein by reference in its entirety.

Certain embodiments disclosed herein include a method of manufacturing asubstantially planar LLEC or LLEF system, the method comprising joiningwith a substrate multiple first reservoirs wherein selected ones of themultiple first reservoirs include a reducing agent, and wherein firstreservoir surfaces of selected ones of the multiple first reservoirs areproximate to a first substrate surface; and joining with the substratemultiple second reservoirs wherein selected ones of the multiple secondreservoirs include an oxidizing agent, and wherein second reservoirsurfaces of selected ones of the multiple second reservoirs areproximate to the first substrate surface, wherein joining the multiplefirst reservoirs and joining the multiple second reservoirs comprisesjoining using tattooing. In embodiments the substrate can comprisegauzes comprising dots or electrodes.

Further embodiments can include a method of manufacturing a LLEC or LLEFsystem, the method comprising joining with a substrate multiple firstreservoirs wherein selected ones of the multiple first reservoirsinclude a reducing agent, and wherein first reservoir surfaces ofselected ones of the multiple first reservoirs are proximate to a firstsubstrate surface; and joining with the substrate multiple secondreservoirs wherein selected ones of the multiple second reservoirsinclude an oxidizing agent, and wherein second reservoir surfaces ofselected ones of the multiple second reservoirs are proximate to thefirst substrate surface, wherein joining the multiple first reservoirsand joining the multiple second reservoirs comprises: combining themultiple first reservoirs, the multiple second reservoirs, and multipleparallel insulators to produce a pattern repeat arranged in a firstdirection across a plane, the pattern repeat including a sequence of afirst one of the parallel insulators, one of the multiple firstreservoirs, a second one of the parallel insulators, and one of themultiple second reservoirs; and weaving multiple transverse insulatorsthrough the first parallel insulator, the one first reservoir, thesecond parallel insulator, and the one second reservoir in a seconddirection across the plane to produce a woven apparatus.

Embodiments disclosed herein include LLEC and LLEF systems that canproduce an electrical stimulus and/or can electromotivate,electroconduct, electroinduct, electrotransport, and/or electrophoreseone or more therapeutic materials in areas of target tissue (e.g.,iontophoresis), and/or can cause one or more biologic or other materialsin proximity to, on or within target tissue to be rejuvenated. Furtherdisclosure relating to materials that can produce an electrical stimuluscan be found in U.S. Pat. No. 7,662,176 entitled FOOTWEAR APPARATUS ANDMETHODS OF MANUFACTURE AND USE issued Feb. 16, 2010, which isincorporated herein by reference in its entirety.

In embodiments “ink” or “paint” can comprise any conductive solutionsuitable for forming an electrode on a surface, such as a conductivemetal solution. In embodiments “printing” or “painted” can comprise anymethod of applying a conductive material such as a conductive liquidmaterial to a material upon which a matrix is desired, such as a fabric.

In embodiments printing devices can be used to produce LLEC or LLEFsystems disclosed herein. For example, inkjet or “3D” printers can beused to produce embodiments.

In certain embodiments the binders or inks used to produce LLEC or LLEFsystems disclosed herein can include, for example, poly cellulose inks,poly acrylic inks, poly urethane inks, silicone inks, and the like. Inembodiments the type of ink used can determine the release rate ofelectrons from the reservoirs. In embodiments various materials can beadded to the ink or binder such as, for example, conductive or resistivematerials can be added to alter the shape or strength of the electricfield. Other materials, such as silicon, can be added to enhance scarreduction. Such materials can also be added to the spaces betweenreservoirs.

In embodiments, fabric embodiments disclosed herein can be woven of atleast two types of fibers; fibers comprising sections treated or coatedwith a substance capable of forming a positive electrode; and fiberscomprising sections treated or coated with a substance capable offorming a negative electrode. The fabric can further comprise fibersthat do not form an electrode. Long lengths of fibers can be woventogether to form fabrics. For example, the fibers can be woven togetherto form a regular pattern of positive and negative electrodes.

Embodiments disclosed herein include a multilayer fabric, for example alayer that can produce an LLEC/LLEF as described herein, a hydrationlayer, and a waterproof layer.

LLEC/LLEF Systems, Devices, and Methods of Use

In embodiments, methods and devices disclosed herein can be used for topre-treat areas where surgery is to be performed. Methods disclosedherein can include applying a disclosed embodiment to an area to betreated. Embodiments can include selecting or identifying a patient inneed of treatment. In embodiments, methods disclosed herein can includeapplication of an antibacterial agent to an area to be treated. Incertain embodiments, disclosed methods include application of anantibacterial to a device disclosed herein.

In embodiments, disclosed methods include application to the treatmentarea or the device of an antibacterial. In embodiments the antibacterialcan be, for example, alcohols, aldehydes, halogen-releasing compounds,peroxides, anilides, biguanides, bisphenols, halophenols, heavy metals,phenols and cresols, quaternary ammonium compounds, and the like. Inembodiments the antibacterial agent can comprise, for example, ethanol,isopropanol, glutaraldehyde, formaldehyde, chlorine compounds, iodinecompounds, hydrogen peroxide, ozone, peracetic acid, formaldehyde,ethylene oxide, triclocarban, chlorhexidine, alexidine, polymericbiguanides, triclosan, hexachlorophene, PCMX (p-chloro-m-xylenol),silver compounds, mercury compounds, phenol, cresol, cetrimide,benzalkonium chloride, cetylpyridinium chloride, ceftolozane/tazobactam,ceftazidime/avibactam, ceftaroline/avibactam, imipenem/MK-7655,plazomicin, eravacycline, brilacidin, and the like.

In embodiments, disclosed methods include application to the treatmentarea of a cosmetic agent. Examples of suitable cosmetic agents include,but are not limited to: inorganic sunscreens such as titanium dioxideand zinc oxide; organic sunscreens such as octyl-methoxy cinnamates;retinoids; dimethylaminoathanol (DMAE), copper containing peptides,vitamins such as vitamin E, vitamin A, vitamin C, and vitamin B andvitamin salts or derivatives such as ascorbic acid di-glucoside andvitamin E acetate or palmitate; alpha hydroxy acids and their precursorssuch as glycolic acid, citric acid, lactic acid, malic acid, mandelicacid, ascorbic acid, alpha-hydroxybutyric acid, alpha-hydroxyisobutyricacid, alpha-hydroxyisocaproic acid, atrrolactic acid,alpha-hydroxyisovaleric acid, ethyl pyruvate, galacturonic acid,glucoheptonic acid, glucoheptono 1,4-lactone, gluconic acid,gluconolactone, glucuronic acid, glucuronolactone, isopropyl pyruvate,methyl pyruvate, mucic acid, pyruvic acid, saccharic acid, saccaric acid1,4-lactone, tartaric acid, and tartronic acid; beta hydroxy acids suchas beta-hydroxybutyric acid, beta-phenyl-lactic acid, andbeta-phenylpyruvic acid; tetrahydroxypropyl ethylene-diamine,N,N,N′,N′-Tetrakis(2-hydroxypropyl)ethylenediamine (THPED); andbotanical extracts such as green tea, soy, milk thistle, algae, aloe,angelica, bitter orange, coffee, goldthread, grapefruit, hoellen,honeysuckle, Job's tears, lithospermum, mulberry, peony, puerarua, nice,and safflower; and salts and prodrugs thereof

In embodiments, the cosmetic agent contains a depigmentation agent.Examples of suitable depigmentation agents include, but are not limitedto: soy extract; soy isoflavones; retinoids such as retinol; kojic acid;kojic dipalmitate; hydroquinone; arbutin; transexamic acid; vitaminssuch as niacin and vitamin C; azelaic acid; linolenic acid and linoleicacid; placertia; licorice; and extracts such as chamomile and green tea;and salts and prodrugs and combinations thereof.

In embodiments, compounds that modify resistance to commonantibacterials can be employed. For example, some resistance-modifyingagents may inhibit multidrug resistance mechanisms, such as drug effluxfrom the cell, thus increasing the susceptibility of bacteria to anantibacterial. In embodiments, these compounds can includePhe-Arg-β-naphthylamide, or β-lactamase inhibitors such as clavulanicacid and sulbactam.

In an exemplary embodiment, a method disclosed herein comprises applyinga conductive antibacterial to an area where treatment is desired, thenapplying over the agent a bioelectric device that comprises amulti-array matrix of biocompatible microcells.

Certain embodiments include LLEC or LLEF systems comprising embodimentsdesigned to be used on irregular, non-planar, or “stretching” surfaces.Embodiments disclosed herein can be used with numerous irregularsurfaces of the mouth, including the gingiva, hard palate, soft palate,molars, premolars, canine, incisors, frenulum of upper lip, frenulum oflower lip, superior vestibule, inferior vestibule, tongue, fauces, etc.Additional embodiments disclosed herein can be used in areas wheretissue is prone to movement, for example the inner cheek, upper andlower lip, etc.

In certain embodiments, the substrate can be shaped to fit a particularregion of the oral cavity. Embodiments include methods and devices fortreating oral diseases in a patient in the need thereof. This method cancomprise a oral cavity device for example a mouth guard, a mouth tray, adental retainer, dental bridges, dentures, or the like with a LLEC andLLEF system attached, printed, connected, etched, implanted or the likeon the surface or subsurface.

In an embodiment, methods and devices disclosed herein can be used totreat oral conditions in a patient in need thereof. Examples of suchtreatments include the treatment of stomatitis (e.g., canker sores, andcold sores); periodontitis (e.g., aggressive periodontitis, chronicperiodontitis, peridontitis as a manifestation of systemic disease, ornecrotizing periodontal disease); gingivitis (e.g., acute gingivitis, orchronic gingivitis); tooth decay (e.g., smooth surface, or pit andfissure, or root); apthous ulcer (e.g., minor ulcers, or major ulcers,or herpetiform ulcers); systemic lupus erythematosus; and bleedingdisorders.

In embodiments, methods and devices disclosed herein can be used afteran oral surgery on a patient in the need thereof, for example,endodontic surgery (e.g., root canal, or pulpotomy, or pulpectomy, orapicoectomy); prosthodontics (e.g., implants, or veneers, or bridges, ordentures, or implant-supported prosthesis); orthodontic treatment (e.g.,implants and implant-supported prosthesis, or extraction, orfiberotomy); periodontics treatment (e.g., gum recession, or gum graft,or dental implant, or scaling and root planing, or chronicperiodontitis); oral and maxillofacial procedure (e.g., dentoalveolar,osseointegrated dental implants, or cheek augmentation, or lipenhancement); cleft pallet procedures (e.g., cleft lip repair, or repairsoft palate, or repair hard palate, or bone grafting of the jaw, or anyfurther oral corrections).

Embodiments disclosed herein can comprise a method for killing bacteriaand then repairing damaged tissue for a patient in the need thereof. Forexample, embodiments can comprise a gauze pad, absorbent beads, mouthguard, mouth tray, or the like that can be pressed up or be in contactwith the gums, teeth, or other parts of the oral cavity. In embodimentsdisclosed herein the bacteria can be for example, Staphylococcusepidermidis, Staphylococcus aureus, Streptococcus mitis, Streptococcussalivarius, Streptococcus mutans, Enterococcus faecalis, Bacteroidessp., Lactobacillus sp., fungus, yeast, viruses, or the like.

Embodiments disclosed herein can comprise dental products. For example,embodiments can comprise a gum or tooth cream wherein the gum or toothcream is applied on the gums or on and between the teeth and theelectrode surface. Embodiments disclosed herein can comprise an oralprocedure. For example, embodiments can be employed before, after, orduring an oral procedure, such as before, after, or during a gum graft,tooth cleaning, or tooth extraction. Certain embodiments can compriseuse of a device disclosed herein before, after, or during a resurfacingprocedure.

In embodiments, the oral product disclosed herein can comprise ananti-plaque active agent and anti-tartar agent. Examples of anti-plaqueand anti-tartar agents include, but are not limited to fluoride,xylitol, triclosan/copolymer, stannous fluoride, triclosan/copolymer,pyropohosphate, hexametaphosphate, zinc, chlorine dioxide, zincchloride, potassium nitrate, potassium citrate, potassium chloride,stannous fluoride, strantium chloride, calcium carbonate, silicas,magnesium carbonate, aluminum oxide, and argonite.

In an exemplary embodiment, a method disclosed herein comprises applyinga conductive oral product to an area where treatment is desired, thenapplying over the oral product a bioelectric device that comprises amulti-array matrix of biocompatible microcells. An embodiment asdisclosed can comprise the dental tray shown in FIG. 15 . Deviceinterior 150 comprises a multi-array matrix of biocompatible microcells.Such matrices can include a first array comprising a pattern ofmicrocells, for example formed from a first conductive solution, thesolution including a metal species; and a second array comprising apattern of microcells, for example formed from a second conductivesolution, the solution including a metal species capable of defining atleast one voltaic cell for spontaneously generating at least oneelectrical current with the metal species of the first array when saidfirst and second arrays are introduced to an electrolytic solution andsaid first and second arrays are not in physical contact with eachother. Device exterior 155 can comprise a smooth surface to maximizeuser comfort. In embodiments, the device comprises discontinuousregions.

Embodiments disclosed herein comprise catheters and catheter dressingscomprising a multi-array matrix of biocompatible microcells. Suchmatrices can include a first array comprising a pattern of microcells,for example formed from a first conductive solution, the solutionincluding a metal species; and a second array comprising a pattern ofmicrocells, for example formed from a second conductive solution, thesolution including a metal species capable of defining at least onevoltaic cell for spontaneously generating at least one electricalcurrent with the metal species of the first array when said first andsecond arrays are introduced to an electrolytic solution and said firstand second arrays are not in physical contact with each other.

As depicted in FIG. 16 , a catheter dressing can include a layer 165formed to shape around a catheter. The layer can comprise any suitablematerial, for example a thermoformable polymer. The catheter dressingfurther comprises one or more layers comprising the multi-array matrixof biocompatible microcells 160. In embodiments, the dressing is held inplace with an additional bandage, for example a Tegaderm 1626W. Inembodiments the various layers are joined, for example with an adhesive.In embodiments, the one or more layers comprising the biocompatiblemicrocells comprise an adhesive. In embodiments, the one or more layerscomprising the biocompatible microcells are clear or transparent.

FIG. 17 shows an additional embodiment comprising a catheter insertionor puncture site 170, as well as a “slit” 177 in the dressing to aid indressing application and removal. The catheter dressing furthercomprises one or more layers comprising the multi-array matrix ofbiocompatible microcells 175. In embodiments, the dressing is held inplace with an additional bandage, for example a Tegaderm 1626 W.

In embodiments, the one or more layers comprising the multi-array matrixof biocompatible microcells can be, for example, less than 1 mm thick, 1mm thick, more than 1 mm thick, more than 2 mm thick, more than 3 mmthick, more than 4 mm thick, more than 5 mm thick, more than 6 mm thick,more than 7 mm thick, more than 8 mm thick, more than 9 mm thick, morethan 10 mm thick, more than 15 mm thick, more than 20 mm thick, morethan 30 mm thick, more than 40 mm thick, more than 50 mm thick, or thelike.

In embodiments, catheter dressings disclosed herein can be used for anysuitable clinical application. For example, the disclosed catheterdressings can be used for IV sites, PICC lines, Hickman catheter sites,and the like.

EXAMPLES

The following non-limiting examples are provided for illustrativepurposes only in order to facilitate a more complete understanding ofrepresentative embodiments. These examples should not be construed tolimit any of the embodiments described in the present specification.

Example 1 Cell Migration Assay

The in vitro scratch assay is an easy, low-cost and well-developedmethod to measure cell migration in vitro. The basic steps involvecreating a “scratch” in a cell monolayer, capturing images at thebeginning and at regular intervals during cell migration to close thescratch, and comparing the images to quantify the migration rate of thecells. Compared to other methods, the in vitro scratch assay isparticularly suitable for studies on the effects of cell-matrix andcell-cell interactions on cell migration, mimic cell migration duringwound healing in vivo and are compatible with imaging of live cellsduring migration to monitor intracellular events if desired. In additionto monitoring migration of homogenous cell populations, this method hasalso been adopted to measure migration of individual cells in theleading edge of the scratch.

Human keratinocytes were plated under plated under placebo or a LLECsystem described herein (labeled “PROCELLERA®”). Cells were also platedunder silver-only or zinc-only dressings. After 24 hours, the scratchassay was performed. Cells plated under the PROCELLERA® device displayedincreased migration into the “scratched” area as compared to any of thezinc, silver, or placebo dressings. After 9 hours, the cells platedunder the PROCELLERA® device had almost “closed” the scratch. Thisdemonstrates the importance of electrical activity to cell migration andinfiltration.

In addition to the scratch test, genetic expression was tested.Increased insulin growth factor (IGF)-1 R phosphorylation wasdemonstrated by the cells plated under the PROCELLERA® device ascompared to cells plated under insulin growth factor alone.

Integrin accumulation also affects cell migration. An increase inintegrin accumulation was achieved with the LLEC system. Integrin isnecessary for cell migration, and is found on the leading edge ofmigrating cell.

Thus, the tested LLEC system enhanced cellular migration and IGF-1R/integrin involvement. This involvement demonstrates the effect thatthe LLEC system had upon cell receptors involved with the wound healingprocess.

Example 2 Wound Care Study

The medical histories of patients who received “standard-of-care” woundtreatment (“SOC”; n=20), or treatment with a LLEC device as disclosedherein (n=18), were reviewed. The wound care device used in the presentstudy consisted of a discrete matrix of silver and zinc dots. Asustained voltage of approximately 0.8 V was generated between the dots.The electric field generated at the device surface was measured to be0.2-1.0 V, 10-50 μA.

Wounds were assessed until closed or healed. The number of days to woundclosure and the rate of wound volume reduction were compared. Patientstreated with LLEC received one application of the device each week, ormore frequently in the presence of excessive wound exudate, inconjunction with appropriate wound care management. The LLEC was keptmoist by saturating with normal saline or conductive hydrogel.Adjunctive therapies (such as negative pressure wound therapy [NPWT],etc.) were administered with SOC or with the use of LLEC unlesscontraindicated. The SOC group received the standard of care appropriateto the wound, for example antimicrobial dressings, barrier creams,alginates, silver dressings, absorptive foam dressings, hydrogel,enzymatic debridement ointment, NPWT, etc. Etiology-specific care wasadministered on a case-by-case basis. Dressings were applied at weeklyintervals or more. The SOC and LLEC groups did not differ significantlyin gender, age, wound types or the length, width, and area of theirwounds.

Wound dimensions were recorded at the beginning of the treatment, aswell as interim and final patient visits. Wound dimensions, includinglength (L), width (W) and depth (D) were measured, with depth measuredat the deepest point. Wound closure progression was also documentedthrough digital photography. Determining the area of the wound wasperformed using the length and width measurements of the wound surfacearea.

Closure was defined as 100% epithelialization with visible effacement ofthe wound. Wounds were assessed 1 week post-closure to ensure continuedprogress toward healing during its maturation and remodeling phase.

Wound types included in this study were diverse in etiology anddimensions, thus the time to heal for wounds was distributed over a widerange (9-124 days for SOC, and 3-44 days for the LLEC group).Additionally, the patients often had multiple co-morbidities, includingdiabetes, renal disease, and hypertension. The average number of days towound closure was 36.25 (SD=28.89) for the SOC group and 19.78(SD=14.45) for the LLEC group, p=0.036. On average, the wounds in theLLEC treatment group attained closure 45.43% earlier than those in theSOC group.

Based on the volume calculated, some wounds improved persistently whileothers first increased in size before improving. The SOC and the LLECgroups were compared to each other in terms of the number of instanceswhen the dimensions of the patient wounds increased (i.e., woundtreatment outcome degraded). In the SOC group, 10 wounds (50% for n=20)became larger during at least one measurement interval, whereas 3 wounds(16.7% for n=18) became larger in the LLEC group (p=0.018). Overall,wounds in both groups responded positively. Response to treatment wasobserved to be slower during the initial phase, but was observed toimprove as time progressed.

The LLEC wound treatment group demonstrated on average a 45.4% fasterclosure rate as compared to the SOC group. Wounds receiving SOC weremore likely to follow a “waxing-and-waning” progression in wound closurecompared to wounds in the LLEC treatment group.

Compared to localized SOC treatments for wounds, the LLEC (1) reduceswound closure time, (2) has a steeper wound closure trajectory, and (3)has a more robust wound healing trend with fewer incidence of increasedwound dimensions during the course of healing.

Example 3 LLEC Influence on Human Keratinocyte Migration

An LLEC-generated electrical field was mapped, leading to theobservation that LLEC generates hydrogen peroxide, known to drive redoxsignaling. LLEC-induced phosphorylation of redox-sensitive IGF-1 R wasdirectly implicated in cell migration. The LLEC also increasedkeratinocyte mitochondrial membrane potential.

The LLEC was made of polyester printed with dissimilar elemental metals.It comprises alternating circular regions of silver and zinc dots, alongwith a proprietary, biocompatible binder added to lock the electrodes tothe surface of a flexible substrate in a pattern of discrete reservoirs.When the LLEC contacts an aqueous solution, the silver positiveelectrode (cathode) is reduced while the zinc negative electrode (anode)is oxidized. The LLEC used herein consisted of metals placed inproximity of about 1 mm to each other thus forming a redox couple andgenerating an ideal potential on the order of 1 Volt. The calculatedvalues of the electric field from the LLEC were consistent with themagnitudes that are typically applied (1-10 V/cm) in classicalelectrotaxis experiments, suggesting that cell migration observed withthe bioelectric dressing is likely due to electrotaxis.

Measurement of the potential difference between adjacent zinc and silverdots when the LLEC is in contact with de-ionized water yielded a valueof about 0.2 Volts. Though the potential difference between zinc andsilver dots can be measured, non-intrusive measurement of the electricfield arising from contact between the LLEC and liquid medium wasdifficult. Keratinocyte migration was accelerated by exposure to anAg/Zn LLEC. Replacing the Ag/Zn redox couple with Ag or Zn alone did notreproduce the effect of keratinocyte acceleration.

Exposing keratinocytes to an LLEC for 24 h significantly increased greenfluorescence in the dichlorofluorescein (DCF) assay indicatinggeneration of reactive oxygen species under the effect of the LLEC. Todetermine whether H₂O₂ is generated specifically, keratinocytes werecultured with a LLEC or placebo for 24 h and then loaded with PF6-AM(Peroxyfluor-6 acetoxymethyl ester; an indicator of endogenous H₂O₂).Greater intracellular fluorescence was observed in the LLECkeratinocytes compared to the cells grown with placebo. Over-expressionof catalase (an enzyme that breaks down H₂O₂) attenuated the increasedmigration triggered by the LLEC. Treating keratinocytes with N-AcetylCysteine (which blocks oxidant-induced signaling) also failed toreproduce the increased migration observed with LLEC. Thus, H₂O₂signaling mediated the increase of keratinocyte migration under theeffect of the electrical stimulus.

External electrical stimulus can up-regulate the TCA (tricarboxylicacid) cycle. The stimulated TCA cycle is then expected to generate moreNADH and FADH₂ to enter into the electron transport chain and elevatethe mitochondrial membrane potential (Am). Fluorescent dyes JC-1 andTMRM were used to measure mitochondrial membrane potential. JC-1 is alipophilic dye which produces a red fluorescence with high Am and greenfluorescence when Am is low. TMRM produces a red fluorescenceproportional to Am. Treatment of keratinocytes with LLEC for 24 hdemonstrated significantly high red fluorescence with both JC-1 andTMRM, indicating an increase in mitochondrial membrane potential andenergized mitochondria under the effect of the LLEC. As a potentialconsequence of a stimulated TCA cycle, available pyruvate (the primarysubstrate for the TCA cycle) is depleted resulting in an enhanced rateof glycolysis. This can lead to an increase in glucose uptake in orderto push the glycolytic pathway forward. The rate of glucose uptake inHaCaT cells treated with LLEC was examined next. More than two foldenhancement of basal glucose uptake was observed after treatment withLLEC for 24 h as compared to placebo control.

Keratinocyte migration is known to involve phosphorylation of a numberof receptor tyrosine kinases (RTKs). To determine which RTKs areactivated as a result of LLEC, scratch assay was performed onkeratinocytes treated with LLEC or placebo for 24 h. Samples werecollected after 3 h and an antibody array that allows simultaneousassessment of the phosphorylation status of 42 RTKs was used to quantifyRTK phosphorylation. It was determined that LLEC significantly inducesIGF-1 R phosphorylation. Sandwich ELISA using an antibody againstphospho-IGF-1 R and total IGF-1 R verified this determination. Asobserved with the RTK array screening, potent induction inphosphorylation of IGF-1 R was observed 3 h post scratch under theinfluence of LLEC. IGF-1 R inhibitor attenuated the increasedkeratinocyte migration observed with LLEC treatment.

MBB (monobromobimane) alkylates thiol groups, displacing the bromine andadding a fluorescent tag (lamda emission=478 nm). MCB (monochlorobimane)reacts with only low molecular weight thiols such as glutathione.Fluorescence emission from UV laser-excited keratinocytes loaded witheither MBB or MCB was determined for 30 min. Mean fluorescence collectedfrom 10,000 cells showed a significant shift of MBB fluorescenceemission from cells. No significant change in MCB fluorescence wasobserved, indicating a change in total protein thiol but notglutathione. HaCaT cells were treated with LLEC for 24 h followed by ascratch assay. Integrin expression was observed by immuno-cytochemistryat different time points. Higher integrin expression was observed 6 hpost scratch at the migrating edge.

Consistent with evidence that cell migration requires H₂O₂ sensing, wedetermined that by blocking H₂O₂ signaling by decomposition of H₂O₂ bycatalase or ROS scavenger, N-acetyl cysteine, the increase inLLEC-driven cell migration is prevented. The observation that the LLECincreases H₂O₂ production is significant because in addition to cellmigration, hydrogen peroxide generated in the wound margin tissue isrequired to recruit neutrophils and other leukocytes to the wound,regulates monocyte function, and VEGF signaling pathway and tissuevascularization. Therefore, external electrical stimulation can be usedas an effective strategy to deliver low levels of hydrogen peroxide overtime to mimic the environment of the healing wound and thus should helpimprove wound outcomes. Another phenomenon observed duringre-epithelialization is increased expression of the integrin subunitalpha-v. There is evidence that integrin, a major extracellular matrixreceptor, polarizes in response to applied ES and thus controlsdirectional cell migration. It may be noted that there are a number ofintegrin subunits, however we chose integrin av because of evidence ofassociation of alpha-v integrin with IGF-1 R, modulation of IGF-1receptor signaling, and of driving keratinocyte locomotion.Additionally, integrin alpha v has been reported to contain vicinalthiols that provide site for redox activation of function of theseintegrins and therefore the increase in protein thiols that we observeunder the effect of ES may be the driving force behind increasedintegrin mediated cell migration. Other possible integrins which may beplaying a role in LLEC-induced IGF-1 R mediated keratinocyte migrationare a5 integrin and a6 integrin.

Materials and Methods

Cell culture—Immortalized HaCaT human keratinocytes were grown inDulbecco's low-glucose modified Eagle's medium (Life Technologies,Gaithersburg, Md., U.S.A.) supplemented with 10% fetal bovine serum, 100U/ml penicillin, and 100 μg/ml streptomycin. The cells were maintainedin a standard culture incubator with humidified air containing 5% CO2 at37° C.

Scratch assay—A cell migration assay was performed using culture inserts(IBIDI®, Verona, Wis.) according to the manufacturers instructions. Cellmigration was measured using time-lapse phase-contrast microscopyfollowing withdrawal of the insert. Images were analyzed using theAxioVision Rel 4.8 software.

N-Acetyl Cysteine Treatment—Cells were pretreated with 5 mM of the thiolantioxidant N-acetylcysteine (Sigma) for 1 h before start of the scratchassay.

IGF-1 R inhibition—When applicable, cells were preincubated with 50 nMIGF-1 R inhibitor, picropodophyllin (Calbiochem, MA) just prior to theScratch Assay.

Cellular H₂O₂ Analysis—To determine intracellular H₂O₂ levels, HaCaTcells were incubated with 5 pM PF6-AM in PBS for 20 min at roomtemperature. After loading, cells were washed twice to remove excess dyeand visualized using a Zeiss Axiovert 200M microscope.

Catalase gene delivery—HaCaT cells were transfected with 2.3×107 pfuAdCatalase or with the empty vector as control in 750 μl of media.Subsequently, 750 μl of additional media was added 4 h later and thecells were incubated for 72 h.

RTK Phosphorylation Assay—Human Phospho-Receptor Tyrosine Kinasephosphorylation was measured using Phospho-RTK Array kit (R & DSystems).

ELISA-Phosphorylated and total IGF-1 R were measured using a DuoSet ICELISA kit from R&D Systems.

Determination of Mitochondrial Membrane Potential—Mitochondrial membranepotential was measured in HaCaT cells exposed to the LLEC or placebousing TMRM or JC-1 (MitoProbe JC-1 Assay Kit for Flow Cytometry, LifeTechnologies), per manufacturers instructions for flow cytometry.

Integrin alpha V Expression—Human HaCaT cells were grown under the MCDor placebo and harvested 6 h after removing the IBIDI® insert. Stainingwas done using antibody against integrin aV (Abeam, Cambridge, Mass.).

Example 4 Induction of Pre-Angiogenic Responses in Vascular EndothelialCells by Signaling Through VEGF Receptors

Materials and Methods

Cell Cultures and Reagents

Tissue culture reagents were obtained from Life Technologies UK. TheVEGFR inhibitor (catalog number 676475), the PI3K inhibitor LY294002(catalog number 440202), the Akt inhibitor (catalog number 124005) andthe Rho kinase inhibitor Y27632 (catalog number 688001) were allobtained from Calbiochem. Rhodamine-phalloidin (E3478) was obtained fromMolecular Probes (Leiden, The Netherlands) and anti-tubulin conjugatedwith FITC was obtained from Sigma. The HUVEC cell line from ATCC wasused prior to passage 10. Dulbecco's modified Eagle's medium (DMEM) with10% fetal bovine serum (FBS) was used for culture cells and EF exposureexperiments.

Electric Field (EF) Stimulation

HUVEC cells were seeded in a trough formed by two parallel (1 cm apart)strips of glass coverslip (No. 1, length of 22 mm) fixed to the base ofthe dish with silicone grease. Scratch lines were made perpendicular tothe long axis of the chamber with a fine sterile needle and used asreference marks for directed cell migration. Cells were incubated for24-48 hours (37° C., 5% CO₂) before a roof coverslip was applied andsealed with silicone grease. The final dimensions of the chamber,through which current was passed, were 22×10×0.2 mm. Agar-salt bridgesnot less than 15 cm long were used to connect silver/silver-chlorideelectrodes in beakers of Steinberg's solution (58 mM NaCl, 0.67 mM KCl,0.44 mM Ca(NO₃)₂, 1.3 mM MgSO₄, 4.6 mM Trizma base, pH 7.8-8.0), topools of excess culture medium at either side of the chamber. Fieldstrengths were measured directly at the beginning of, the end of andduring each experiment. No fluctuations in field strength were observed.For drug inhibition experiments, cells were incubated with the VEGFRinhibitor 4-[(4′-chloro-2′-fluoro)phenylamino]-6,7-dimethoxyquinazoline(50 μM), the PI3K inhibitor LY294002 (50 μM), an Akt inhibitor1-L-6-hydroxymethyl-chiro-inositol2-[(R)-2-O-methyl-3-O-octadecylcarbonate] (50 μM), the Rho kinaseinhibitor Y27632 (50 μM), both Akt and Rho kinase inhibitors (10 μMeach) or latrunculin (50 nM) for 1 hour before EF stimulation. The sameconcentration of drug was present during EF exposure in a CO₂ incubator.

Quantification of Cell Behavior

A series of images was taken with an image analyser immediately beforeEF exposure and at 4, 8 and 24 hours of EF exposure. Cell orientationwas quantified as an orientation index (Oi), which is defined as Oi=cos2(α), where α is the angle formed by the long axis of a cell with a linedrawn perpendicular to the field lines. A cell with its long axisparallel to the vector of the EF will have an Oi of −1, and a cell withits long axis exactly perpendicular to the EF vector will have an Oi of+1. A randomly oriented population of cells will have an average Oi{defined as [Σ_(n) cos 2(α)]÷n} of 0. The significance of thistwo-dimensional orientation distribution against randomness wascalculated using Rayleigh's distribution. A long:short axis ratio wascalculated for assessment of elongation.

Mean migration rate and directedness were quantified over 4 hoursbecause cells multiplied during longer EF exposures, making it difficultto define a clear migration path. The angle (θ) that each cell movedwith respect to the imposed EF vector was measured. The cos(θ)(directedness) is +1, if the cell moved directly along the field linestoward the cathode, 0 if the cell moved perpendicular to the EF vectorand −1 if the cell moved directly towards the positive pole. Averagingthe cosines {[Σ_(i) cos(θ)]÷N, where N is the total number of cells}yields an average directedness of cell movement.

A commercially available VEGF165 ELISA kit was obtained from R and D(Minneapolis, Minn.), and the detailed technical instructions werefollowed. Confocal microscopy was as described. Statistical analyseswere performed using unpaired, two-tailed Student's t-test. Data areexpressed as mean±s.e.m.

Results

Cells cultured without exposure to the EF had the typical cobblestonemorphology, with the long axis of the cell body oriented randomly. Incontrast, endothelial cells cultured in DC EFs underwent areorientation, with their long axis coming to lie perpendicular to thevector of the applied EF. This elongation and alignment in an applied EFresembles the response of endothelial cells to fluid shear stress.

Cell alignment was quantified using an orientation index Oi=cos 2(α),where α is the angle formed between the long axis of a cell and a linedrawn perpendicular to the field lines. In cells oriented perpendicularto the field vector, the Oi is +1, cells parallel to the field vectorgive an Oi of −1 and random orientation gives an Oi of 0. We comparedthe elongation and reorientation of single cells with those of cells inmonolayers. They were broadly similar, with single cells respondingquicker and showing a significantly higher Oi (0.56±0.04, n=245) at 4hours of EF exposure than cells in a monolayer sheet (0.35±0.03, n=528).Both single cells and cells in monolayers, however, had a similar Oi by8 hours (0.71±0.03, n=227 and 0.62±0.03, n=312, respectively).

The perpendicular orientation of endothelial cells showed both time andvoltage dependency. Significant orientation was observed as early as 4hours after the onset of the EF. A steady increase of Oi indicatesgradually increasing perpendicular orientation with continued exposure.Longer EF exposure, up to 3 days at 100 mV mm⁻¹ (1 mV across a cell 10μm wide), induced striking orientation and elongation. EF exposure didnot induce any detrimental effects on the cells, which were perfectlyhealthy for up to 3-4 days in EFs.

Voltage dependency was more obvious at later times, with a higher Oi forcells cultured at higher voltages. After 24 hours at 300 mV mm⁻¹, almostall the cells were perpendicular. An EF strength as low as 75 mV mm⁻¹induced significant perpendicular orientation, with Oi of 0.19(significantly different from random orientation, p=4.4×10⁻⁶, n=433),whereas an EF of 50 mV mm⁻¹ did not. The threshold field strengthinducing perpendicular orientation of the endothelial cells wastherefore between 50 mV mm⁻¹ and 75 mV mm⁻¹. This is low, representingonly 0.5-0.75 mV across a cell with a diameter of 10 μm.

Reorientation of endothelial cells in EFs requires VEGFR activation

VEGF activation is a pivotal elements in angiogenic responses andenhanced angiogenesis by electric stimulation in vivo is mediatedthrough VEGFR activation. To test whether EF-induced endothelial cellorientation might involve VEGF signaling, we quantified levels of VEGF.EF exposure (200 mV mm⁻¹, the same as that measured at skin wounds)significantly enhanced levels of VEGF released into the culture medium.Marked elevation of VEGF in the culture medium was observed as early as5 minutes after onset of the EF; this was reduced at 1 hour and 2 hours,rose again at 4 hours, and reached a high level by 24 hours.

Inhibition of VEGFR activation by inhibiting both VEGFR-1 and VEGFR-2with the drug4-[(4′-chloro-2′-fluoro)phenylamino]-6,7-dimethoxyquinazoline completelyabolished the reorientation of cells in an EF. This drug is a potentVEGFR inhibitor that inhibits the receptor tyrosine kinase activity (50%inhibitory concentrations of 2.0 μM and 100 nM for VEGFR-1 and VEGFR-2,respectively). It is very selective for VEGFR-1 and VEGFR-2 tyrosinekinase activity compared with that associated with the epidermal growthfactor (EGF) receptor (50-fold and 3800-fold, respectively). Themorphology of the cells treated with VEGFR inhibitor was very similar tocontrol cells. Cells still elongated, although their long axis wasslightly reduced, but they were oriented randomly. Inhibition of VEGFRscould conceivably have had detrimental effects on the long-termviability of cells and this could have influenced their orientationresponses. To test for this, we compared the orientation response aftera short period of inhibitor and EF application. The orientation responsewas completely abolished at 4 hours and 8 hours in an EF after VEGFRinhibition. The Oi values of the cells treated with VEGFR inhibitor were−0.16±0.05 and −0.05±0.05 in EF for 4 hours and 8 hours, respectively,which is significantly different from the non-inhibitor-treated valuesof 0.36±0.05 and 0.53±0.05 (P<0.01).

Reorientation of Endothelial Cells Involved the PI3K-Akt Pathway

VEGFR activation lead to endothelial cell migration, cell survival andproliferation, which require the activation of Akt, a downstreameffectors of PI3K. Both the PI3K inhibitor LY294002 (50 μM) and the Aktinhibitor (50 μM) significantly decreased the orientation response.

The concentration of either drug alone would be expected to inhibit PI3Kand Akt activation completely but neither drug inhibited perpendicularreorientation completely, and significant Oi values remained, indicatingthat other signaling mechanisms must be involved.

Role of Rho and Integrin in EF-Induced Reorientation of EndothelialCells

The Rho family of GTPases regulates VEGF-stimulated endothelial cellmotility and reorganization of the actin cytoskeleton, which areimportant in endothelial cell retraction and in the formation ofintercellular gaps. The Rho kinase inhibitor, Y27632, decreased theorientation response significantly, with Oi values of 0.55±0.05,0.45±0.05 and 0.24±0.05 at 10 μM, 20 μM and 50 μM, respectively.Significant Oi values nonetheless remained even at 50 μM, indicatingthat multiple signaling mechanisms must be involved.Mitogen-activated-protein kinase inhibition with U0126 (50 μM), likeY27632 (0.33±0.03), decreased the orientation to a similar extent.

Because both Akt and Rho kinase inhibitors individually showed partialinhibition, perhaps the two enzymes function in different pathways toinduce cell reorientation. To test this, a combination of the twoinhibitors was used. The orientation response was abolished completelyby using Akt and Rho kinase inhibitors together (both at 10 μM)(Oi=−0.10±0.06; compared to control=0.80±0.09, P<0.0001) (FIG. 3B).

Integrins, especially αvβ3, are important in endothelial cell movementand alignment to shear stress and mechanical stimulation. HUVEC cellswere incubated with a blocking antibody against αvβ3(LM609) (20 μg ml⁻¹)for 1 hour and then exposed to an EF (200 mV mm⁻¹) with the antibodypresent. Blocking αvβ3 had no effect on orientation to the EF, cellsreoriented normally (Oi=0.72±0.03, n=110, compared with thecontrol=0.80±0.09, n=124, P>0.05).

Small EFs Elongated Endothelial Cells

HUVEC cells elongated dramatically in an EF. By contrast, cells culturedwith no EF retained a more-cobblestone-like appearance. Striking cellelongation was induced by a voltage drop of about 0.7-4.0 mV across acell of ˜15 μm in diameter. We quantified the elongation of the cellsusing a long:short axis ratio. A perfectly round cell has a long:shortaxis ratio of 1 and, as cells elongate, the ratio increases. Controlcells (no EF) showed no increase in long:short axis over 24 hours inculture. Elongation responses were both time and voltage dependent. Thelong:short axis ratio of EF exposed cells indicated gradual cellelongation throughout the 24 hour experimental period. The voltagedependency of the elongation response was more obvious at later times,with a greater long:short axis ratio for cells cultured at higher EFs.The threshold for EF-induced endothelial cell elongation was between50-75 mV mm⁻¹, again 0.5-0.75 mV across a cell 10 μm in diameter. Theelongation response of endothelial cells was more marked than that seenpreviously at the same EF strengths, in corneal and lens epithelialcells.

VEGFR, PI3K-Akt and Rho signaling are involved in the elongationresponse

The signaling elements required for reorientation are also involved inelongation, but there are subtle differences. The VEGFR inhibitor (50μM) had no effect on the long:short axis ratio of control cells butsignificantly decreased the long:short axis ratio in EF-treated cells(P<0.002). Both the PI3K inhibitor LY294002 and the Akt inhibitor alsosignificantly decreased the long:short axis ratio (both P<0.0001 versuscontrol). Cells treated with these drugs elongated less, with LY294002the more effective in suppressing EF-induced elongation. The Rho kinaseinhibitor, Y27632 also significantly decreased the long:short axis ratio(P<0.0001, FIG. 5B), whereas the αvβ3-blocking antibody significantlyinhibited the elongation response (3.12±0.008 compared with the control3.65±0.15, P=0.007).

Cytoskeleton alignment and the consequence of actin filament disruption

To control changes in cell shape, reorientation and migration,extracellular stimuli initiate intracellular signaling that modifiescytoskeletal organization. Both actin filaments and microtubules werealigned in the direction of cell elongation. Latrunculin A, a toxininhibiting actin polymerization, completely abolished the EF-inducedelongation response and suppressed the orientation responsesignificantly (P<0.001) but not fully.

Small EFs direct migration of endothelial cells towards the anode

Endothelial cells migrated directionally toward the anode when culturedin EFs. The directional migration was slow but steady during the EFexposure and was more evident for single cells than for sheets of cells.Cells migrated directionally towards the anode while elongating andreorienting perpendicularly. Lamellipodial extension toward the anodewas marked. Directional migration was obvious at a physiological EFstrength of 100 mV mm⁻¹. The threshold field strength that could inducedirectional migration was therefore below 100 mV mm⁻¹. Cell migrationwas quantified as previously and significant anodal migration wasevident (P<0.0001). Migration speed, however, remained constant beforeand after EF exposure, at 1-2 μm hour⁻¹, which is significantly slowerthan most other cell types migrating in an EF.

Example 5 Generation of Superoxide

A LLEC system was tested to determine the effects on superoxide levelswhich can activate signal pathways. PROCELLERA® LLEC system increasedcellular protein sulfhydryl levels. Further, the PROCELLERA® systemincreased cellular glucose uptake in human keratinocytes. Increasedglucose uptake can result in greater mitochondrial activity and thusincreased glucose utilization, providing more energy for cellularmigration and proliferation. This can “prime” the wound healing processbefore a surgical incision is made and thus speed incision healing.

Example 6 Effect on Propionibacterium acnes

Bacterial Strains and Culture

The main bacterial strain used in this study is Propionibacterium acnesand multiple antibiotics-resistant P. acnes isolates are to beevaluated.

ATCC medium (7 Actinomyces broth) (BD) and/or ATCC medium (593 choppedmeat medium) is used for culturing P. acnes under an anaerobic conditionat 37° C. All experiments are performed under anaerobic conditions.

Culture

LNA (Leeming-Notman agar) medium is prepared and cultured at 34° C. for14 days.

Planktonic Cells

P. acnes is a relatively slow-growing, typically aero-tolerantanaerobic, Gram-positive bacterium (rod). P. acnes is cultured underanaerobic condition to determine for efficacy of an embodiment disclosedherein (PROCELLERA®). Overnight bacterial cultures are diluted withfresh culture medium supplemented with 0.1% sodium thioglycolate in PBSto10⁵ colony forming units (CFUs). Next, the bacterial suspensions (0.5mL of about 105) are applied directly on PROCELLERA® (2″×2″) and controlfabrics in Petri-dishes under anaerobic conditions. After 0 h and 24 hpost treatments at 37° C., portions of the sample fabrics are placedinto anaerobic diluents and vigorously shaken by vortexing for 2 min.The suspensions are diluted serially and plated onto anaerobic platesunder an anaerobic condition. After 24 h incubation, the survivingcolonies are counted. The LLEC limits bacterial proliferation.

Example 7 Pre-Treatment Prior to Spinal Fusion Surgery

Prior to spinal fusion surgery, the patient wears a three-layer vestthat covers only the back as shown in FIG. 10 . The vest consists of alayer of standard PROCELLERA® backed by a hydration layer of polyvinylalcohol fibers fabricated into a uniform, spongelike open cell porestructure. An outer layer of a waterproof polyester fabric surrounds thehydration layer. A compression shirt is worn over the vest to provideintimate contact between the electrodes and the skin. The vest is soakedin a warm water/salt solution prior to placement on the patient for thenight. The material remains hydrated and produces voltage for over 24hours (see FIGS. 11 and 12 ). The material is cool to the touch but doesnot feel wet. A dry shirt can be worn over the vest.

The LLEC initiates the incision-healing process by; 1) reducing oreliminating microorganism presence around the incision site; 2)increasing integrin accumulation; 3) increasing cellular proteinsulfhydryl levels; 4) increasing H₂O₂ production; and 5) up-regulatingthe TCA (tricarboxylic acid) cycle.

Example 8 Pre-Treatment Prior to Blepharoplasty

The patient is scheduled to undergo blepharoplasty in 1 week. A mask asseen in FIG. 9 is made with printed electrodes as described herein,using the pattern described in FIG. 1 . Prior to donning the mask, thepatient applies a conductive agent to the skin around his eyes. The maskand conductive agent are reapplied to the patient's skin each night.After 1 week of nightly wear, the incision-healing process is initiatedand accelerated.

Example 9 Pre-Treatment Prior to Brow Lift

The patient is scheduled to undergo a brow lift in 2 week. A mask asseen in FIG. 9 is made with printed electrodes as described herein,using the pattern described in FIG. 1 . Prior to donning the mask, thepatient applies a conductive agent to the skin around his brow. The maskand conductive agent are reapplied to the patient's skin each night.After 2 weeks of nightly wear, the incision-healing process is initiatedand accelerated.

Example 10 Pre-Treatment Prior to Rhinoplasty

The patient is scheduled to undergo rhinoplasty in 1 week. A device withprinted electrodes as described herein is shaped to fit over and aroundthe patient's nose. Prior to donning the device, the patient applies aconductive agent to the skin on and around her nose. The mask andconductive agent are reapplied to the patient's skin each night. After 1week of nightly wear, the incision-healing process is initiated andaccelerated.

Example 11 Pre-Treatment Prior to Rotator Cuff Surgery

A patient is scheduled to undergo rotator cuff surgery. A device withprinted electrodes and void regions (as shown in FIG. 8A) to aid infitting around the patient's shoulder is prepared. Prior to donning thedevice, the patient applies a conductive agent to the skin on and aroundhis shoulder. The device and conductive agent are reapplied to thepatient's skin each night. After wearing the device for 24 hours priorto the surgery (the conductive agent is reapplied every 6 hours), theincision-healing process is initiated and accelerated.

Example 12 Making an Electroceutical Fabric

An electroceutical fabric is produced by weaving fibers wherein sectionsof the fibers are coated or treated with materials capable of producingelectricity and forming a battery in the presence of an electrolyte.

The fabric is woven of three types of fibers; fibers comprising discretesections spray coated with silver forming a positive electrode; fiberscomprising discrete sections spray coated with zinc forming a negativeelectrode; and fibers that do not form an electrode. The individualfibers are shown in FIG. 13 . Silver deposits 130 form microbatterieswith zinc deposits 137 when in common contact with a conductivematerial.

The fabric is seen in FIG. 14 . Silver deposits 140 form microbatterieswith zinc deposits 145 when in common contact with a conductivematerial.

Example 13 Modulation of Bacterial Gene Expression and Enzyme Activity

Treatment of biofilms presents a major challenge, because bacterialiving within them enjoy increased protection against host immuneresponses and are markedly more tolerant to antibiotics. Bacteriaresiding within biofilms are encapsulated in an extracellular matrix,consisting of several components including polysaccharides, proteins andDNA which acts as a diffusion barrier between embedded bacteria and theenvironment thus retarding penetration of antibacterial agents.Additionally, due to limited nutrient accessibility, thebiofilm-residing bacteria are in a physiological state of low metabolismand dormancy increasing their resistance towards antibiotic agents.

Chronic wounds present an increasing socio-economic problem and anestimated 1-2% of western population suffers from chronic ulcers andapproximately 2-4% of the national healthcare budget in developedcountries is spent on treatment and complications due to chronic wounds.The incidence of non-healing wounds is expected to rise as a naturalconsequence of longer lifespan and progressive changes in lifestyle likeobesity, diabetes, and cardiovascular disease. Non-healing skin ulcersare often infected by biofilms. Multiple bacterial species reside inchronic wounds; with Pseudomonas aeruginosa, especially in largerwounds, being the most common. P. aeruginosa is suspected to delayhealing of leg ulcers. Also, surgical success with split graft skintransplantation and overall healing rate of chronic venous ulcers ispresumably reduced when there is clinical infection by P. aeruginosa.

P. aeruginosa biofilm is often associated with chronic wound infection.The BED (“BED” or “bioelectric device” or PROCELLERA® as disclosedherein) consists of a matrix of silver-zinc coupled biocompatiblemicrocells, which in the presence of conductive wound exudate activatesto generate an electric field (0.3-0.9V). Growth (measured as O.D andcfu) of pathogenic Pseudomonas aeruginosa strain PAO1 in LB media wasmarkedly arrested in the presence of the BED (p<0.05, n=4). PAO1 biofilmwas developed in vitro using a polycarbonate filter model. Grownovernight in LB medium at 37° C. bacteria were cultured on sterilepolycarbonate membrane filters placed on LB agar plates and allowed toform a mature biofilm for 48 h. The biofilm was then exposed to BED orplacebo for the following 24 h. Structural characterization usingscanning electron microscopy demonstrated that the BED markedlydisrupted biofilm integrity as compared to no significant effectobserved using a commercial silver dressing commonly used for woundcare. Staining of extracellular polymeric substance, PAO1 staining, anda vital stain demonstrated a decrease in biofilm thickness and number oflive bacterial cells in the presence of BED (n=4). BED repressed theexpression of quorum sensing genes lasR and rhlR (p<0.05, n=3). BED wasalso found to generate micromolar amounts of superoxide (n=3), which areknown reductants and repress genes of the redox sensing multidrug effluxsystem mexAB and mexEF (n=3, p<0.05). BED also down-regulated theactivity of glycerol-3-phosphate dehydrogenase, an electric fieldsensitive enzyme responsible for bacterial respiration, glycolysis, andphospholipid biosynthesis (p<0.05, n=3).

Materials and Methods

In-Vitro Biofilm Model

PAO1 biofilm was developed in vitro using a polycarbonate filter model.Cells were grown overnight in LB medium at 37° C. bacteria were culturedon sterile polycarbonate membrane filters placed on LB agar plates andallowed to form a mature biofilm for 48 h. The biofilm was then exposedto BED or placebo for the following 24 h.

Energy Dispersive X-ray Spectroscopy (EDS)

EDS elemental analysis of the Ag/ZN BED was performed in anenvironmental scanning electron microscope (ESEM, FEI XL-30) at 25 kV. Athin layer of carbon was evaporated onto the surface of the dressing toincrease the conductivity.

Scanning Electron Microscopy

Biofilm was grown on circular membranes and was then fixed in a 4%formaldehyde/2% glutaraldehyde solution for 48 hours at 4° C., washedwith phosphate-buffered saline solution buffer, dehydrated in a gradedethanol series, critical point dried, and mounted on an aluminum stub.The samples were then sputter coated with platinum (Pt) and imaged withthe SEM operating at 5 kV in the secondary electron mode (XL 30S; FEG,FEI Co., Hillsboro, Oreg.).

Live/Dead Staining

The LIVE/DEAD BacLight Bacterial Viability Kit for microscopy andquantitative assays was used to monitor the viability of bacterialpopulations. Cells with a compromised membrane that are considered to bedead or dying stain red, whereas cells with an intact membrane staingreen.

EPR Spectroscopy

EPR measurements were performed at room temperature using a Bruker ER300 EPR spectrometer operating at X-band with a TM 110 cavity. Themicrowave frequency was measured with an EIP Model 575 source-lockingmicrowave counter (EIP Microwave, Inc., San Jose, Calif.). Theinstrument settings used in the spin trapping experiments were asfollows: modulation amplitude, 0.32 G; time constant, 0.16 s; scan time,60 s; modulation frequency, 100 kHz; microwave power, 20 mW; microwavefrequency, 9.76 GHz. The samples were placed in a quartz EPR flat cell,and spectra were recorded at ambient temperature (25° C.). Serial 1-minEPR acquisitions were performed. The components of the spectra wereidentified, simulated, and quantitated as reported. The double integralsof DEPMPO experimental spectra were compared with those of a 1 mM TEMPOsample measured under identical settings to estimate the concentrationof superoxide adduct.

Quantification of mRNA and miRNA Expression

Total RNA, including the miRNA fraction, was isolated using Norgen RNAisolation kit, according to the manufacturers protocol. Gene expressionlevels were quantified with real-time PCR system and SYBR Green (AppliedBiosystems) and normalized to nadB and proC as housekeeping genes.Expression levels were quantified employing the 2 (−ΔΔct) relativequantification method.

Glycerol-3-Phosphate Dehydrogenase Assay

The glycerol-3-phosphate dehydrogenase assay was performed using anassay kit from Biovision, Inc. following manufacturer's instructions.Briefly, cells (˜1×10⁶) were homogenized with 200 μl ice cold GPDH Assaybuffer for 10 minutes on ice and the supernatant was used to measureO.D. and GPDH activity calculated from the results.

Statistics

Control and treated samples were compared by paired t test. Student's ttest was used for all other comparison of difference between means.P<0.05 was considered significant.

Ag/Zn BED Disrupts P. aeruginosa Biofilm

To validate the chemical composition of the dressing, we collected highresolution electron micrographs using an environmental scanning electronmicroscope. Our element maps indicate that silver particles areconcentrated in the golden dots of the polyester cloth, while zincparticles are concentrated in the grey dots.

As illustrated in FIG. 18A, P. aeruginosa was grown in round bottomtubes in LB medium with continuous shaking and absorbance was measuredby calculating optical density at 600 nm at different time points. Itwas observed that Ag/Zn BED and the control dressing with equal amountof silver inhibited bacterial growth (n=4) (FIG. 18B,C). When bacteriais grown in an agar plate with Ag/Zn BED dressing or placebo embedded inthe agar, the zone of inhibition is clearly visible in the case of Ag/ZnBED thus demonstrating its bacteriostatic property, while placebo withsilver dressing showed a smaller zone of inhibition, indicating theeffect role of electric field as compared to topical contact. (FIG.18D). However, as evident from scanning electron microscope images (FIG.19 ); EPS staining (FIG. 20 ); and live/dead staining (FIG. 21 ), Ag/ZnBED disrupts biofilm much better while silver does not have any effecton biofilm disruption. Silver has been recognized for its antimicrobialproperties for centuries. Most studies on the antibacterial efficacy ofsilver, with particular emphasis on wound healing, have been performedon planktonic bacteria. Silver ions, bind to and react with proteins andenzymes, thereby causing structural changes in the bacterial cell walland membranes, leading to cellular disintegration and death of thebacterium. Silver also binds to bacterial DNA and RNA, therebyinhibiting the basal life processes.

Silver is effective against mature biofilms of P. aeruginosa, but onlyat a high silver concentration. A concentration of 5-10 μg/mL silversulfadiazine has been reported to eradicate biofilm whereas a lowerconcentration (1 μg/mL) had no effect. Therefore, the concentration ofsilver in currently available wound dressings is much too low fortreatment of chronic biofilm wounds. FIG. 18 shows PAO1 staining of thebiofilm demonstrating the lack of elevated mushroom like structures inthe Ag/Zn BED treated sample.

Ag/Zn BED Down-Regulates Quorum Sensing Genes

The pathogenicity of P. aeruginosa is attributable to an arsenal ofvirulence factors. The production of many of these extracellularvirulence factors occurs only when the bacterial cell density hasreached a threshold (quorum). Quorum sensing is controlled primarily bytwo cell-to-cell signaling systems, called las and rhl, which are bothcomposed of a transcriptional regulator (LasR and RhlR, respectively)and an autoinducer synthase (Lasl and RhII, respectively). In P.aeruginosa, Lasl produces 3OC12-HSL. LasR, then, responds to this signaland the LasR:3OC12-HSL complex activates transcription of many genesincluding rhlR, which encodes a second quorum sensing receptor, RhlRwhich binds to autoinducer C4-HSL produced by RhII. RhlR:C4-HSL alsodirects a large regulon of genes. P. aeruginosa defective in QS iscompromised in their ability to form biofilms. Quorum sensing inhibitorsincrease the susceptibility of the biofilms to multiple types ofantibiotics.

To test the effect of the electric field on quorum sensing genes, wesubjected the mature biofilm to the Ag/Zen BED or placebo for 12 hoursand looked at gene expression levels. We selected an earlier time point,because by 24 hours, as in earlier experiments, most bacteria underAg/Zn BED treatment were dead. We found a significant down regulation oflasR and rhlR (n=4, p<0.05). lasR transcription has been reported toweakly correlate with the transcription of lasA, lasB, toxA and aprA. Wedid not, however, find any significant difference in their expressionlevels at this time point, although we found them down regulated in theAg/Zn BED treated samples at the 24 hour time point (data not shown).(FIG. 23 ).

Ag/Zn BED Represses the Redox Sensing Multidrug Efflux System in P.aeruginosa

Ag/Zn BED acts as a reducing agent and reduces protein thiols. Oneelectron reduction of dioxygen O₂, results in the production ofsuperoxide anion. Molecular oxygen (dioxygen) contains two unpairedelectrons. The addition of a second electron fills one of its twodegenerate molecular orbitals, generating a charged ionic species withsingle unpaired electrons that exhibit paramagnetism. Superoxide anion,which can act as a biological reductant and can reduce disulfide bonds,is finally converted to hydrogen peroxide is known to have bactericidalproperties. Here, we used electron paramagnetic resonance (EPR) todetect superoxide directly upon exposure to the bioelectric dressing.Superoxide spin trap was carried out using DEPMPO(2-(diethoxyphosphoryl)-2-methyl-3,4-dihydro-2H-pyrrole 1- oxide) and ˜1μM superoxide anion production was detected upon 40 mins of exposure toAg/Zn BED (FIG. 24 ). MexR and MexT are two multidrug efflux regulatorsin P. aeruginosa which uses an oxidation-sensing mechanism. Oxidation ofboth MexR and MexT results in formation of intermolecular disulfidebonds, which activates them, leading to dissociation from promoter DNAand de-repression of MexAB-oprM and MexEF-oprN respectively, while in areduced state, they do not transcribe the operons. Induction of Mexoperons leads not only to increased antibiotic resistance but also torepression of the quorum sensing cascades and several virulence factors.We observe down-regulation of the downstream Mex genes MexA, MexB, MexEand MexF (but not MexC and MexD) (n=4, p<0.05), in Ag/Zn BED treatedsamples, inactive forms of MexR and MexT in their reduced states. Toconfirm the reducing activity of the Ag/Zn BED, the experiments wererepeated with 10 mM DTT and similar results were observed. (FIG. 25 ).

Ag/Zn BED Diminishes Glycerol-3-Phosphate Dehydrogenase Enzyme Activity

Electric fields can affect molecular charge distributions on manyenzymes. Glycerol-3-phosphate dehydrogenase is an enzyme involved inrespiration, glycolysis, and phospholipid biosynthesis and is expectedto be influenced by external electric fields in P. aeruginosa. Weobserved significantly diminished glycerol-3-phosphate dehydrogenaseenzyme activity by treating P. aeruginosa biofilm to the Ag/Zn BED for12 hours (n=3). (FIG. 26 ).

Example 14 LLEC Influence on Biofilm Properties

In this study ten clinical wound pathogens associated with chronic woundinfections were used for evaluating the anti-biofilm properties of aLLEC. Hydrogel and drip-flow reactor (DFR) biofilm models were employedfor the efficacy evaluation of the wound dressing in inhibitingbiofilms. Biofilms formed with Acinetobacter baumannii, Corynebacteriumamycolatum, Escherichia coli, Enterobacter aerogenes, Enterococcusfaecalis CI 4413, Klebsiella pneumonia, Pseudomonas aeruginosa, Serratiamarcescens, Staphylococcus aureus, and Streptococcus equi clinicalisolates were evaluated. For antimicrobial susceptibility testing ofbiofilms, 10⁵ CFU/mL bacteria was used in both biofilm models. Forpoloxamer hydrogel model, the LLECs (25 mm diameter) were applieddirectly onto the bacterial biofilm developed onto 30% poloxamerhydrogel and Muller-Hinton agar plates, and incubated at 37° C. for 24 hto observe any growth inhibition. In the DFR biofilm model, bacteriawere deposited onto polycarbonate membrane as abiotic surface, andsample dressings were applied onto the membrane. The DFR biofilm wasincubated in diluted trypticase soy broth (TSB) at room temperature for72 h. Biofilm formations were evaluated by crystal violet staining underlight microscopy, and anti-biofilm efficacy was demonstrated byreduction in bacterial numbers.

Example 15 Modulation of Mammalian Gene Expression and Enzyme Activity

Grown overnight in LB medium at 37° C., primary human dermal fibroblastsare cultured on sterile polycarbonate membrane filters placed on LB agarplates for 48 h. The cells are then exposed to BED or placebo for thefollowing 24 h. BED represses the expression of glyceraldehyde3-phosphate dehydrogenase. BED also down-regulates the activity ofglyceraldehyde 3-phosphate dehydrogenase.

Example 16 Modulation of Insect Gene Expression and Enzyme Activity

Grown overnight in LB medium at 37° C., drosophila S2 cells are culturedon sterile polycarbonate membrane filters placed on LB agar plates for48 h. The cells are then exposed to BED or placebo for the following 24h. BED represses the expression of insect P450 enzymes. BED alsodown-regulates the activity of insect P450 enzymes.

In closing, it is to be understood that although aspects of the presentspecification are highlighted by referring to specific embodiments, oneskilled in the art will readily appreciate that these disclosedembodiments are only illustrative of the principles of the subjectmatter disclosed herein. Therefore, it should be understood that thedisclosed subject matter is in no way limited to a particularmethodology, protocol, and/or reagent, etc., described herein. As such,various modifications or changes to or alternative configurations of thedisclosed subject matter can be made in accordance with the teachingsherein without departing from the spirit of the present specification.Lastly, the terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to limit the scope ofthe present disclosure, which is defined solely by the claims.Accordingly, embodiments of the present disclosure are not limited tothose precisely as shown and described.

Certain embodiments are described herein, including the best mode knownto the inventor for carrying out the methods and devices describedherein. Of course, variations on these described embodiments will becomeapparent to those of ordinary skill in the art upon reading theforegoing description. Accordingly, this disclosure includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described embodiments in all possiblevariations thereof is encompassed by the disclosure unless otherwiseindicated herein or otherwise clearly contradicted by context.

Groupings of alternative embodiments, elements, or steps of the presentdisclosure are not to be construed as limitations. Each group member maybe referred to and claimed individually or in any combination with othergroup members disclosed herein. It is anticipated that one or moremembers of a group may be included in, or deleted from, a group forreasons of convenience and/or patentability. When any such inclusion ordeletion occurs, the specification is deemed to contain the group asmodified thus fulfilling the written description of all Markush groupsused in the appended claims.

Unless otherwise indicated, all numbers expressing a characteristic,item, quantity, parameter, property, term, and so forth used in thepresent specification and claims are to be understood as being modifiedin all instances by the term “about.” As used herein, the term “about”means that the characteristic, item, quantity, parameter, property, orterm so qualified encompasses a range of plus or minus ten percent aboveand below the value of the stated characteristic, item, quantity,parameter, property, or term. Accordingly, unless indicated to thecontrary, the numerical parameters set forth in the specification andattached claims are approximations that may vary. At the very least, andnot as an attempt to limit the application of the doctrine ofequivalents to the scope of the claims, each numerical indication shouldat least be construed in light of the number of reported significantdigits and by applying ordinary rounding techniques. Notwithstandingthat the numerical ranges and values setting forth the broad scope ofthe disclosure are approximations, the numerical ranges and values setforth in the specific examples are reported as precisely as possible.Any numerical range or value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Recitation of numerical ranges ofvalues herein is merely intended to serve as a shorthand method ofreferring individually to each separate numerical value falling withinthe range. Unless otherwise indicated herein, each individual value of anumerical range is incorporated into the present specification as if itwere individually recited herein.

The terms “a,” “an,” “the” and similar referents used in the context ofdescribing the disclosure (especially in the context of the followingclaims) are to be construed to cover both the singular and the plural,unless otherwise indicated herein or clearly contradicted by context.All methods described herein can be performed in any suitable orderunless otherwise indicated herein or otherwise clearly contradicted bycontext. The use of any and all examples, or exemplary language (e.g.,“such as”) provided herein is intended merely to better illuminate thedisclosure and does not pose a limitation on the scope otherwiseclaimed. No language in the present specification should be construed asindicating any non-claimed element essential to the practice ofembodiments disclosed herein.

Specific embodiments disclosed herein may be further limited in theclaims using consisting of or consisting essentially of language. Whenused in the claims, whether as filed or added per amendment, thetransition term “consisting of” excludes any element, step, oringredient not specified in the claims. The transition term “consistingessentially of” limits the scope of a claim to the specified materialsor steps and those that do not materially affect the basic and novelcharacteristic(s). Embodiments of the present disclosure so claimed areinherently or expressly described and enabled herein.

1. A catheter comprising one or more biocompatible electrodes configuredto generate at least one of a low level electric field (LLEF) or lowlevel electric current (LLEC).
 2. The catheter of claim 1 wherein thebiocompatible electrodes comprise a first array comprising a firstpattern of microcells formed from a first conductive material, and asecond array comprising a second pattern of microcells formed from asecond conductive material.
 3. The catheter of claim 2 wherein the firstconductive material and the second conductive material comprise the samematerial.
 4. The catheter of claim 3 wherein the first array and thesecond array each comprise a discrete circuit.
 5. The catheter of claim4, further comprising a power source.
 6. The catheter of claim 2 whereinthe first array and the second array spontaneously generate a LLEF. 7.The catheter of claim 6 wherein the first array and the second arrayspontaneously generate a LLEC when contacted with an electrolyticsolution.
 8. A method for catheterizing a patient comprising use one ormore biocompatible electrodes configured to generate at least one of alow level electric field (LLEF) or low level electric current (LLEC). 9.The method of claim 8 wherein the biocompatible electrodes comprise afirst array comprising a first pattern of microcells formed from a firstconductive material, and a second array comprising a second pattern ofmicrocells formed from a second conductive material.
 10. The method ofclaim 9 wherein the first conductive material and the second conductivematerial comprise the same material.
 11. The method of claim 9 whereinthe first array and the second array each comprise a discrete circuit.12. The method of claim 4, wherein said catheter further comprises apower source.
 13. The method of claim 9 wherein the first array and thesecond array spontaneously generate a LLEF.
 14. The method of claim 13wherein the first array and the second array spontaneously generate aLLEC when contacted with an electrolytic solution.